Compositions and methods for controling carbohydrate and fat metabolism

ABSTRACT

The subject of the invention is a composition comprising at least a mixture of molecules obtained at least from:
           Chrysanthellum indicum , and     Cynara scolymus , and     Vaccinium myrtillus,    
said mixture of molecules also comprising piperine.
       

     This composition is particularly useful as a nutritional product or health product for preventing and/or combating carbohydrate and/or fat metabolism disorders in humans and animals.

TECHNICAL FIELD

The present invention relates to the prevention and treatment ofcarbohydrate and/or fat metabolism disorders in humans and animals.

BACKGROUND OF THE INVENTION

Type 2 diabetes, the most common form of diabetes, is a chronic,progressive metabolic disease. It is characterized by chronichyperglycemia, i.e. an abnormally high concentration of sugar in theblood, and intolerance to carbohydrates. The main cause of this chronichyperglycemic state is resistance to insulin and inadequate insulinsecretion in response to a given metabolic state, but other factors maybe involved, as described in Ismail-Beigi F N Engl J Med 2012;366:1319-27. The main approaches for combating type 2 diabetes should beto reduce the levels of glycemia, to lower the glycated hemoglobin(HbA₁c) to a level of less than or equal to 6.5%, and to improve thesensitivity to insulin.

The treatment of type 2 diabetes consists firstly of a change inlifestyle or hygieno-dietetic measures (HDM: the diet, smoking, physicaland sports activities). If the HDMs are insufficient, then use should bemade of antidiabetic therapeutic agents, generally metformin. However,metformin has many medical contraindications, such as chronic renalinsufficiency, acidosis, hypoxia, dehydration, etc. There is thus asubstantial paradox for metformin since its prescription is not possiblein the case of type 2 diabetic patients presenting with renalinsufficiency, but renal insufficiency is one of the common consequencesof type 2 diabetes. Other therapeutic agents have been developed, suchas dipeptidyl peptidase-IV (DPP-IV) inhibitors, glucagon-like peptide-1(GLP-1) analogs, thiazolidinediones (TZDs), sulfonylures, glycosidaseinhibitors (acarbose, miglitol, voglibose), or sodium glucoseco-transporter-2 (SGLT2) inhibitors. However, the correct therapeuticcombination is complex since it requires a large number of factors to betaken into consideration, such as the contraindications and the adverseeffects, which affect the quality of life of the patients and thus theiradherence to the medical treatment. Furthermore, certain therapeuticcombinations increase the all-cause mortality, such as the combinationof metformin with sulfonylures, as described in Prescrire Int 2015;24:130-5.

Since HDMs are very rarely adhered to by patients, it is necessary veryrapidly to establish a therapeutic treatment with all the adverseeffects and contraindications associated with these molecules, and thereis at the present time no solution adapted to the care management oftype 2 diabetic patients between the HDMs and the establishment of atherapeutic treatment.

Moreover, type 2 diabetic patients have a high risk of cardiovascularmorbi-mortality. It is thus also necessary to include in the caremanagement the conventional cardiovascular risk factors especially suchas control of the circulating fats, the weight and the arterialpressure. This necessity currently entails the taking of severalmedicaments of different therapeutic classes simultaneously. However,the combination of drugs may occasionally give rise to serious sidereactions, for instance the simultaneous administration of fibrates andstatins which increases the risk of myopathy, as described in Denke M AJ Manag Care Pharm 2003; 9:17-9.

There is thus a real need for products that can be used both during theattenuated establishment of cardio-metabolic pathologies, characterizedby an increase in certain risk factors (carbohydrate disorders, fatdisorders, excess weight, inflammation, oxidative stress, arterialhypertension), and during the outbreak of these pathologies, especiallytype 2 diabetes.

Moreover, there is an urgent need for preventive solutions andmedicaments whose “multi-target” mechanism of action has advantages interms of compliance, tolerance and efficacy. Such products would make itpossible to reduce the overall risk of cardio-metabolic diseases and toprevent and treat each dysfunction and/or its consequences takenindependently.

SUMMARY OF THE INVENTION

The aim of the invention is to satisfy these various needs by providingcompositions that are capable of acting simultaneously on severalcarbohydrate and fat dysfunctions, which represent both a preventivemeans and a therapeutic means that is advantageous for preventing andtreating cardio-metabolic diseases and complications thereof.

To satisfy its aim, the invention is directed toward compositionscomprising at least a mixture of molecules obtained at least from:

-   -   Chrysanthellum indicum, and    -   Cynara scolymus, and    -   Vaccinium myrtillus,        said mixture of molecules also comprising piperine.

This is a synergistic mixture, the molecules present in the mixture actsynergistically. Extracts of Chrysanthellum indicum, extracts of Cynarascolymus, extracts of Vaccinium myrtillus and piperine have already beendescribed and some of them have been used in nutrition products, but,unexpectedly, the combination of at least one extract of Chrysanthellumindicum, with at least one extract of Cynara scolymus, an extract ofVaccinium myrtillus and piperine leads to surprising results both oncarbohydrate metabolism and on fat metabolism in humans and animals.

To satisfy its aim, the invention is also directed toward compositionsconsisting of at least piperine and particular specific moleculescontained in Chrysanthellum indicum, Cynara scolymus and Vacciniummyrtillus (these molecules being natural and/or synthetic), inparticular a composition comprising a mixture of at least fourmolecules, at least one molecule being piperine, at least one moleculebeing chosen from apigenin 7-O-glucuronide, chrysanthellin A,chrysanthellin B, caffeic acid, luteolin, maritimetin, eriodictyol,isookanin, apigenin, luteolin 7-O-glucoside, maritimein, marein,eriodictyol 7-O-glucoside, flavomarein, apigenin8-C-α-L-arabinoside-6-C-β-D-glucoside (shaftoside), apigenin6,8-C-di-β-D-glucopyranoside (vicenin-2), or analogs thereof, and atleast one molecule being chosen from a dicaffeoylquinic acid, asulfo-monocaffeoylquinic acid, luteolin, luteolin 7-O-glucoside,luteolin 7-O-glucuronide, apigenin 7-O-glucoside, cynaropicrin oranalogs thereof, and at least one molecule being chosen from amonocaffeoylquinic acid, delphinidin 3-galactoside, delphinidin3-glucoside, cyanidin 3-galactoside, delphinidin 3-arabinoside,cyanidine 3-glucoside, petunidin 3-galactoside, cyanidin 3-arabinoside,petunidin 3-glucoside, peonidin 3-galactoside, petunidin 3-arabinoside,peonidin 3-glucoside, malvidin 3-galactoside, malvidin 3-glucoside,malvidin 3-arabinoside, or analogs thereof.

In the present application, the singular or the plural will be usedwithout preference to denote the compositions according to theinvention.

Advantageously, the compositions according to the invention prevent theestablishment of chronic hyperglycemia and decrease glycemia, decreaseglycated hemoglobin, allow an improvement in the tolerance to ingestedcarbohydrates, improve the sensitivity to insulin, but are also capableof acting on other cardiovascular risk factors such as dyslipidemia,excess weight and obesity, and arterial tension. In addition, they havefew or no side effects with regard to those observed with the existingtreatments and those undergoing development.

The invention is thus also directed toward the use of the compositionsas nutritional products or health products, especially as medicaments,in particular for preventing and/or combating carbohydrate and fatmetabolism disorders in humans and animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: the results for four compositions of an oral test of sensitivityto insulin corresponding to the results in table 9 (point II.1);

FIG. 2: the results demonstrating the synergistic effect afforded by acomposition according to the invention, these results corresponding tothe results of table 13 (point II.1) for fasting glycemia;

FIG. 3: the results demonstrating the synergistic effect afforded by acomposition according to the invention, these results corresponding tothe results of table 13 (point II.1) for carbohydrate tolerance;

FIG. 4: the results demonstrating the synergistic effect afforded by acomposition according to the invention, these results corresponding tothe results of table 14 (point II.1) for fasting glycemia;

FIG. 5: the results demonstrating the synergistic effect afforded by acomposition according to the invention, these results corresponding tothe results of table 14 (point II.1) for carbohydrate tolerance;

FIG. 6: the results demonstrating the synergistic effect afforded by acomposition according to the invention, these results corresponding tothe results of table 14 (point II.1) for insulin sensitivity;

FIG. 7: the results for the effect of a composition according to theinvention and metformin, on insulin sensitivity, these resultscorresponding to the results of table 15 (point II.2);

FIG. 8: the results for the effect of a composition according to theinvention on the change in body mass, these results corresponding to theresults of table 15 (point II.2);

FIG. 9: the results for the effect of a composition according to theinvention on the change in fasting glycemia, these results correspondingto the results of table 15 (point II.2);

FIG. 10: the results of western blotting showing an increase in theamount of AMPK in mouse liver with a composition according to theinvention (point II.2);

FIG. 11: the results of tests showing the absence of an acutehypoglycemiant effect with a composition according to the invention(point II.3);

FIG. 12: the results of tests showing the effect of a compositionaccording to the invention on the change in body mass increase (pointII.4).

DETAILED DESCRIPTION OF THE INVENTION

A subject of the invention is a composition comprising at least amixture of molecules obtained at least from:

-   -   Chrysanthellum indicum, and    -   Cynara scolymus, and    -   Vaccinium myrtillus,        said mixture of molecules also comprising piperine.

The invention is thus directed toward a composition comprising at leastpiperine, at least one molecule derived from Chrysanthellum indicum, atleast one molecule derived from Cynara scolymus and at least onemolecule derived from Vaccinium myrtillus.

In addition to Chrysanthellum indicum, Cynara scolymus, Vacciniummyrtillus and piperine, the composition according to the invention mayalso contain other compounds, in particular an extract of Oleo europea.In this case, the composition according to the invention comprises atleast one molecule derived from Chrysanthellum indicum, at least onemolecule derived from Cynara scolymus, at least one molecule derivedfrom Vaccinium myrtillus and at least one molecule derived from Oleoeuropea and piperine.

According to a first embodiment, the composition according to theinvention comprises at least:

-   -   an extract of Chrysanthellum indicum, and    -   an extract of Cynara scolymus, and    -   an extract of Vaccinium myrtillus, and    -   synthetic piperine and/or an extract of Piper containing        piperine.

For the purposes of the invention, the term “extract of a plant “X” orof a plant “X” raw material” means at least one molecule, preferentiallya set of molecules, obtained from the plant “X” via any suitableprocess. Mention may be made in particular of aqueous extracts (obtainedusing an aqueous solvent), alcoholic extracts (obtained using analcoholic solvent) or using an organic solvent, or using a natural fattysubstance or a mixture of natural fatty substances, especially a plantoil or a mixture of plant oils. The term “aqueous solvent” means anysolvent consisting totally or partly of water. Mention may thus be madeof water itself, aqueous-alcoholic solvents in any proportion orsolvents consisting of water and of a compound such as glycerol orpropylene glycol in any proportion. Among the alcoholic solvents,mention may be made especially of ethanol.

For the purposes of the invention, the term “plant or plant rawmaterial” means the whole plant or the plant part, including cellcultures, which has not yet undergone a specific treatment and isintended to be included in the manufacture of a plant preparation.

The plant extracts may be obtained via any suitable process, forexample, via a process comprising the following steps:

-   -   solid/liquid extraction    -   separation/pressing    -   filtration    -   evaporation    -   drying    -   optionally incorporation of additives    -   homogenization    -   conditioning.

The extract of Chrysanthellum indicum is preferably an extract of wholeplant or of the aerial parts.

It may in particular be an aqueous-alcoholic or aqueous or subcriticalCO₂ or subcritical H₂O extract or combined with a heat treatment whichis performed by standard heating or under microwave frequency or underultrasound.

The plant/extract ratio is preferentially between 1/1 and 100/1, inparticular between 1/1 and 25/1.

The composition according to the invention, when intended for Human,preferentially comprises an amount of extract of Chrysanthellum indicumallowing the administration of at least 0.00001 g, in particular between0.00001 g and 0.60 g, of extract of Chrysanthellum indicum per kg ofbody weight of the person to whom the composition is administered andper day.

Preferably, the extract of Chrysanthellum indicum comprises at least onemolecule chosen from apigenin 7-O-glucuronide, chrysanthellin A,chrysanthellin B, caffeic acid, luteolin, maritimetin, eriodictyol,isookanin, apigenin, luteolin 7-O-glucoside, maritimein, marein,eriodictyol 7-O-glucoside, flavomarein, apigenin8-C-α-L-arabinoside-6-C-β-D-glucoside (shaftoside), apigenin6,8-C-di-β-D-glucopyranoside (vicenin-2), or analogs thereof.Preferentially, the extract comprises at least apigenin 7-O-glucuronide.

For the purposes of the present invention, the term “analogs” means allcompounds having a chemical structure similar to another compound, butdiffering therefrom by a certain component. It may differ by one or moreatoms, functional groups, sub-structures, which are replaced with otheratoms, functional groups or sub-structures. Examples that may bementioned include apigenin 7-O-glucuronide analogs such as apigenin7-apioglucoside, apigenin 8-C-glucoside (vitexin), apigenin6-C-glucoside (isovitexin), apigenin 7-O-neohesperidoside, apigenin7-glucoside, apigenin 7-apioglucoside.

The extract of Cynara scolymus is preferably an extract of leaves orroots.

It may in particular be an aqueous-alcoholic or aqueous or subcriticalCO₂ or subcritical H₂O extract or combined with a heat treatment whichis performed by standard heating or under microwave frequency or underultrasound.

The plant/extract ratio is preferentially between 1/1 and 100/1, inparticular between 1/1 and 30/1.

The composition according to the invention, when intended for Human,preferentially comprises an amount of extract of Cynara scolymus thatallows the administration of at least 0.00001 g, in particular between0.00001 g and 0.60 g, of extract of Cynara scolymus per kg of bodyweight of the person to whom the composition is administered and perday. Preferably, the extract of Cynara scolymus comprises at least onemolecule chosen from a dicaffeoylquinic acid, a sulfo-monocaffeoylquinicacid, luteolin, luteolin 7-O-glucoside, luteolin 7-O-glucuronide,apigenin 7-O-glucoside, cynaropicrin, or analogs thereof.Preferentially, the extract comprises at least one dicaffeoylquinicacid.

The extract of Vaccinium myrtillus is preferentially an extract of fruitor leaves.

It may in particular be an aqueous-alcoholic or aqueous or subcriticalCO₂ or subcritical H₂O extract or combined with a heat treatment whichis performed by standard heating or under microwave frequency or underultrasound.

The plant/extract ratio is preferentially between 1/1 and 200/1, inparticular between 1/1 and 60/1.

When it is intended for Human, the composition according to theinvention preferentially comprises an amount of extract of Vacciniummyrtillus allowing the administration of at least 0.00001 g, inparticular between 0.00001 g and 0.60 g, of extract of Vacciniummyrtillus per kg of body weight of the person to whom the composition isadministered and per day. Preferably, the extract of Vaccinium myrtilluscomprises at least one molecule chosen from a monocaffeoylquinic acid,delphinidin 3-galactoside, delphinidin 3-glucoside, cyanidin3-galactoside, delphinidin 3-arabinoside, cyanidin 3-glucoside,petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin 3-glucoside,peonidin 3-galactoside, petunidin 3-arabinoside, peonidin 3-glucoside,malvidin 3-galactoside, malvidin 3-glucoside, malvidin 3-arabinoside, oranalogs thereof. Preferentially, the extract comprises at least onemonocaffeoylquinic acid.

The piperine present in the composition according to the invention maybe contained in an extract of Piper or may be a synthetic piperine.

The topological formula of piperine is as follows:

The composition according to the invention, when intended for Human,preferentially comprises an amount of piperine allowing theadministration of at least 0.001 mg, in particular between 0.001 mg and166 mg, of piperine per kg of body weight of the person to whom thecomposition is administered and per day.

If the piperine is contained in an extract of Piper, the mixture of thecomposition according to the invention comprises said extract. Theextract of Piper is preferentially an extract of Piper nigrum, of Piperaduncum and/or of Piper longum.

The extract of Piper is preferably an extract of fruit or leaves.

It may in particular be an aqueous-alcoholic or aqueous or subcriticalCO₂ or subcritical H₂O extract or combined with a heat treatment that isperformed by standard heating or under microwave frequency or underultrasound.

The plant/extract ratio is preferentially between 1/1 and 10000/1, inparticular between 1/1 and 200/1.

The extract preferentially comprises at least 1% of piperine by weightrelative to the total weight of the extract.

Thus, the mixture of the composition according to the inventionpreferentially comprises:

-   -   at least piperine, and    -   at least one molecule chosen from apigenin 7-O-glucuronide,        chrysanthellin A, chrysanthellin B, caffeic acid, luteolin,        maritimetin, eriodictyol, isookanin, apigenin, luteolin        7-O-glucoside, maritimein, marein, eriodictyol 7-O-glucoside,        flavomarein, apigenin 8-C-α-L-arabinoside-6-C-β-D-glucoside        (shaftoside), apigenin 6,8-C-di-β-D-glucopyranoside (vicenin-2),        or analogs thereof, preferentially apigenin 7-O-glucuronide, and    -   at least one molecule chosen from a dicaffeoylquinic acid, a        sulfo-monocaffeoylquinic acid, luteolin, luteolin 7-O-glucoside,        luteolin 7-O-glucuronide, apigenin 7-O-glucoside, cynaropicrin,        or analogs thereof, preferentially a dicaffeoylquinic acid, and    -   at least one molecule chosen from a monocaffeoylquinic acid,        delphinidin 3-galactoside, delphinidin 3-glucoside, cyanidin        3-galactoside, delphinidin 3-arabinoside, cyanidin 3-glucoside,        petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin        3-glucoside, peonidin 3-galactoside, petunidin 3-arabinoside,        peonidin 3-glucoside, malvidin 3-galactoside, malvidin        3-glucoside, malvidin 3-arabinoside, or analogs thereof,        preferentially at least one monocaffeoylquinic acid.

In addition to the extracts of Chrysanthellum indicum, Cynara scolymusand Vaccinium myrtillus, and piperine, the mixture according to theinvention may also contain other compounds, in particular an extract ofOleo europaea.

The extract of Oleo europaea is preferentially an extract of leaves orfruit.

It may in particular by an aqueous-alcoholic or aqueous or subcriticalCO₂ or subcritical H₂O extract or combined with a heat treatment that isperformed by standard heating or under microwave frequency or underultrasound.

The plant/extract ratio is preferentially between 1/1 and 200/1, inparticular between 1/1 and 60/1.

The composition according to the invention, when intended for Human,preferentially comprises an amount of extract of Oleo europaea allowingthe administration of at least 0.00001 g, in particular between 0.00001g and 0.60 g, of extract of Oleo europaea per kg of body weight of theperson to whom the composition is administered and per day.

Preferably, the extract of Oleo europaea comprises at least one moleculechosen from oleuropein and hydroxytyrosol, or analogs thereof.

By mixture within the meaning of the invention, we mean the combinationof substances (extracts and/or molecules) in solid, liquid or gas thatcan interact chemically or not.

The mixture according to the invention is obtained via any process knownto those skilled in the art. It may be obtained by simple mixing of theconstituents.

Preferentially the ratio of the extract of Chrysanthellumindicum/extract of Cynara scolymus/extract of Vacciniummyrtillus/piperine in the mixture is between 0.01/0.01/0.01/0.0001 and10/10/10/10.

According to one variant, in addition to the mixture consisting ofseveral plant extracts or in place of the mixture consisting of severalplant extracts, the mixture of molecules of the composition according tothe invention may comprise at least:

-   -   a single extract obtained from a mixture of at least two plants        chosen from Chrysanthellum indicum, Cynara scolymus and        Vaccinium myrtillus and Piper and optionally,    -   one extract of Chrysanthellum indicum if the mixture of plants        of the single extract does not comprise Chrysanthellum indicum,    -   one extract of Cynara scolymus if the mixture of plants of the        single extract does not comprise Cynara scolymus,    -   one extract of Vaccinium myrtillus if the mixture of plants of        the single extract does not comprise Vaccinium myrtillus,    -   piperine or an extract of Piper if the mixture of plants of the        single extract does not comprise Piper.

Preferentially, when the composition comprises a single extract, itcomprises:

-   -   a single extract obtained from at least Chrysanthellum indicum,        Cynara scolymus and Vaccinium myrtillus and Piper, and/or    -   piperine and a single extract obtained from at least        Chrysanthellum indicum, Cynara scolymus and Vaccinium myrtillus.

For the purposes of the invention, the term “single extract obtainedfrom several plants “X” or plant “X” raw materials” means a set ofmolecules obtained from a mixture of at least two plants “X” via anysuitable process. Mention may be made in particular of aqueous extracts(obtained using an aqueous solvent), alcoholic extracts (obtained usingan alcoholic solvent) or using an organic solvent, or using a naturalfatty substance or a mixture of natural fatty substances, especially aplant oil or a mixture of plant oils. The term “aqueous solvent” meansany solvent consisting totally or partly of water. Mention may thus bemade of water itself, aqueous-alcoholic solvents in any proportion orsolvents consisting of water and of a compound such as glycerol orpropylene glycol in any proportion. Among the alcoholic solvents,mention may be made especially of ethanol.

For the purposes of the invention, the term “plant or plant rawmaterial” means the whole plant or the plant part, including cellcultures, which has not yet undergone a specific treatment and isintended to be included in the manufacture of a plant preparation.

The single extract of a mixture of plants “X” may be obtained via anysuitable process, for example via a process comprising the followingsteps:

-   -   solid/liquid extraction    -   separation/pressing    -   filtration    -   evaporation    -   drying    -   optionally incorporation of additives    -   homogenization    -   conditioning.

Use is preferentially made of the whole plant or the aerial parts ofChrysanthellum indicum as plant raw material to obtain the singleextract. The single extract is preferentially prepared from at least0.1% of whole plant or of the aerial parts of Chrysanthellum indicum byweight relative to the total weight of the mixture of plants used forpreparing the single extract.

Use is preferentially made of the leaves or roots of Cynara scolymus asplant raw material for obtaining the single extract. The single extractis preferentially prepared from at least 0.1% of leaves or roots ofCynara scolymus by weight relative to the total weight of the mixture ofplants used for preparing the single extract.

Use is preferentially made of the fruit or leaves of Vaccinium myrtillusas plant raw material for obtaining the single extract. The singleextract is preferentially prepared from at least 0.1% of fruit or leavesof Vaccinium myrtillus by weight relative to the total weight of themixture of plants used for preparing the single extract.

If the single extract is obtained from a mixture of plants comprisingPiper, the fruit or leaves of Piper nigrum, of Piper aduncum and/or ofPiper longum are preferentially used as plant raw material for obtainingthe single extract. The single extract is preferentially prepared fromat least 0.0001% of fruit or leaves of Piper nigrum, and/or of Piperaduncum and/or of Piper longum by weight relative to the total weight ofthe mixture of plants used for preparing the single extract.

The single extract may thus comprise, in addition to the othermolecules, at least oleuropein, hydroxytyrosol, and/or at leastpiperine.

Preferably, the single extract comprises:

-   -   at least one molecule chosen from apigenin 7-O-glucuronide,        chrysanthellin A, chrysanthellin B, caffeic acid, luteolin,        maritimetin, eriodictyol, isookanin, apigenin, luteolin        7-O-glucoside, maritimein, marein, eriodictyol 7-O-glucoside,        flavomarein, apigenin 8-C-α-L-arabinoside-6-C-β-D-glucoside        (shaftoside), apigenin 6,8-C-di-β-D-glucopyranoside (vicenin-2),        or analogs thereof, preferentially apigenin 7-O-glucuronide, and    -   at least one molecule chosen from a dicaffeoylquinic acid, a        sulfo-monocaffeoylquinic acid, luteolin, luteolin 7-O-glucoside,        luteolin 7-O-glucuronide, apigenin 7-O-glucoside, cynaropicrin,        or analogs thereof, preferentially a dicaffeoylquinic acid, and    -   at least one molecule chosen from a monocaffeoylquinic acid,        delphinidin 3-galactoside, delphinidin 3-glucoside, cyanidin        3-galactoside, delphinidin 3-arabinoside, cyanidin 3-glucoside,        petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin        3-glucoside, peonidin 3-galactoside, petunidin 3-arabinoside,        peonidin 3-glucoside, malvidin 3-galactoside, malvidin        3-glucoside, malvidin 3-arabinoside, or analogs thereof,        preferentially at least one monocaffeoylquinic acid, and    -   and piperine.

In addition to Chrysanthellum indicum, Cynara scolymus and Vacciniummyrtillus, the single extract of the composition according to theinvention may also preferentially be obtained from Oleo europaea. Use ispreferentially made of the leaves or fruit of Oleo europaea as plant rawmaterial for obtaining the single extract. The single extract ispreferentially prepared from at least 0.1% of leaves or fruit of Oleoeuropaea by weight relative to the total weight of the mixture of plantsused for preparing the single extract.

A particularly suitable embodiment is a composition comprising a singleextract obtained from Chrysanthellum indicum, Cynara scolymus, Vacciniummyrtillus, Piper and Oleo europaea.

The composition according to the invention comprising a single extract,when intended for Human, preferentially comprises an amount of singleextract corresponding to an administration of at least 0.00001 g, inparticular between 0.00001 g and 0.60 g of single extract per kg of bodyweight of the person to whom the composition is administered and perday.

The composition according to the invention comprising plant extractsand/or one or more single extracts thus comprises:

-   -   at least one molecule chosen from apigenin 7-O-glucuronide,        chrysanthellin A, chrysanthellin B, caffeic acid, luteolin,        maritimetin, eriodictyol, isookanin, apigenin, luteolin        7-O-glucoside, maritimein, marein, eriodictyol 7-O-glucoside,        flavomarein, apigenin 8-C-α-L-arabinoside-6-C-β-D-glucoside        (shaftoside), apigenin 6,8-C-di-β-D-glucopyranoside (vicenin-2),        or analogs, preferentially apigenin 7-O-glucuronide, and    -   from a dicaffeoylquinic acid, a sulfo-monocaffeoylquinic acid,        luteolin, luteolin 7-O-glucoside, luteolin 7-O-glucuronide,        apigenin 7-O-glucoside, cynaropicrin, or analogs thereof,        preferentially a dicaffeoylquinic acid, and    -   at least one molecule chosen from a monocaffeoylquinic acid,        delphinidin 3-galactoside, delphinidin 3-glucoside, cyanidin        3-galactoside, delphinidin 3-arabinoside, cyanidin 3-glucoside,        petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin        3-glucoside, peonidin 3-galactoside, petunidin 3-arabinoside,        peonidin 3-glucoside, malvidin 3-galactoside, malvidin        3-glucoside, malvidin 3-arabinoside, or analogs thereof,        preferentially at least one monocaffeoylquinic acid, and    -   at least piperine,    -   and optionally, preferentially at least one molecule chosen from        oleuropein, hydroxytyrosol, or analogs thereof.

According to another aspect, the invention is also directed toward acomposition comprising a mixture of at least four molecules, thesemolecules possibly being synthetic molecules and/or natural molecules,especially derived from plant raw materials:

-   -   at least one molecule being chosen from apigenin        7-O-glucuronide, chrysanthellin A, chrysanthellin B, caffeic        acid, luteolin, maritimetin, eriodictyol, isookanin, apigenin,        luteolin 7-O-glucoside, maritimein, marein, eriodictyol        7-O-glucoside, flavomarein, apigenin        8-C-α-L-arabinoside-6-C-β-D-glucoside (shaftoside), apigenin        6,8-C-di-β-D-glucopyranoside (vicenin-2), or analogs thereof,        and    -   at least one molecule being chosen from a dicaffeoylquinic acid,        a sulfo-monocaffeoylquinic acid, luteolin, luteolin        7-O-glucoside, luteolin 7-O-glucuronide, apigenin 7-O-glucoside,        cynaropicrin, or analogs thereof, and    -   at least one molecule being chosen from a monocaffeoylquinic        acid, delphinidin 3-galactoside, delphinidin 3-glucoside,        cyanidin 3-galactoside, delphinidin 3-arabinoside, cyanidin        3-glucoside, petunidin 3-galactoside, cyanidin 3-arabinoside,        petunidin 3-glucoside, peonidin 3-galactoside, petunidin        3-arabinoside, peonidin 3-glucoside, malvidin 3-galactoside,        malvidin 3-glucoside, malvidin 3-arabinoside, or analogs        thereof, and    -   at least piperine.

At least one of the first three molecules is a synthetic molecule.Indeed, independently of piperine which may be synthetic or natural, atleast one of the other molecules is a synthetic molecule.

In addition to these at least four molecules, the mixture may alsocomprise at least one molecule chosen from oleuropein, hydroxytyrosol,or analogs thereof, these molecules possibly being synthetic or naturalmolecules.

According to a particularly suitable variant, the mixture comprises atleast one dicaffeoylquinic acid, apigenin 7-O-glucuronide, amonocaffeoylquinic acid, piperine and oleuropein. The mixture ofmolecules of the composition according to the invention may consistexclusively of a dicaffeoylquinic acid, apigenin 7-O-glucuronide, amonocaffeoylquinic acid, piperine and oleuropein.

According to other variants, the compositions of the invention maycomprise a mixture of extract(s) and molecule(s) of interest presentedin this application (for example an extract of Chrysanthellum indicumand at least one molecule being chosen from a dicaffeoylquinic acid, asulfo-monocaffeoylquinic acid, luteolin, luteolin 7-O-glucoside,luteolin 7-O-glucuronide, apigenin 7-O-glucoside, cynaropicrin, oranalogs thereof, and at least one molecule being chosen from amonocaffeoylquinic acid, delphinidin 3-galactoside, delphinidin3-glucoside, cyanidin 3-galactoside, delphinidin 3-arabinoside, cyanidin3-glucoside, petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin3-glucoside, peonidin 3-galactoside, petunidin 3-arabinoside, peonidin3-glucoside, malvidin 3-galactoside, malvidin 3-glucoside, malvidin3-arabinoside, or analogs thereof, and at least piperine). Thecompositions according to the invention in their different variants mayconsist exclusively of the elements described (plant extracts and/orsingle extract(s) or mixture of at least four molecules), or else mayalso comprise at least one additional element (products, molecules,extracts, active principles, excipients, etc.) added in addition to theplant extracts and/or the single extract(s) or of the mixture of atleast four molecules, said additional element possibly being chosenfrom:

-   -   the following vitamins: B1, B2, B3, B5, B6, B8, B9, B12 C, A, D,        E, K1 and K2;    -   the following compounds: obeticholic acid, corosolic acid,        polyunsaturated fatty acids of the omega 6 and/or omega 3        family, orotic acid, pangamic acid, para-aminobenzoic acid,        amygdalin, beta-glucans, carnitine, dimethylglycine, imeglimin,        isoflavones, L-arginine, oxytocin, pectin, pyridoxamine,        resveratrol, viniferin, L-citrulline;    -   the following trace elements and minerals: arsenic, boron,        calcium, copper, iron, fluorine, iodine, lithium, manganese,        magnesium, molybdenum, nickel, phosphorus, selenium, vanadium,        zinc;    -   the following microconstituents of non-essential nature:        conjugated linolenic acid, lipoic acid, carotenoids, carnitine,        choline, coenzyme Q10, phytosterols, polyphenols of the tannin        and lignan family, taurine;    -   fructo-oligosaccharides, galacto-oligosaccharides;    -   lactic acid-fermenting bacteria;    -   yeasts, for example red rice yeast (Monascus purpureus);    -   muschroom, for example maitake;    -   products derived from insects that are compatible with the food        and pharmaceutical sector;    -   marijuana and hashish;    -   coating agents: for example hypromellose, microcrystalline        cellulose, stearic acid, talc, sucrose, shellac, povidone,        beeswax;    -   flavors: for example natural flavor of blueberry or natural        flavor of strawberry;    -   acidifying agents such as malic acid;    -   antiagglomerating agents: for example silicon dioxide or        magnesium stearate;    -   thickeners such as xanthan gum, colloidal silica, fatty acid        mono- and diglycerides;    -   stabilizers such as calcium phosphate;    -   emulsifiers such as soybean lecithin;    -   fillers such as corn starch;    -   excipients: for example microcrystalline cellulose, magnesium        stearate or dicalcium phosphate.

The compositions according to the invention may also comprise one ormore extracts of at least one of the following plant raw materialsand/or one or more molecules contained in at least one of the followingplant materials and/or the single extract may also be obtained from atleast one of the following plant raw materials: Abelmoschus esculentus,Abies Alba, Abies balsomea, Abies sibirica, Acacia nilotica, Acaciasenegal, Achillea millefollium, Achyronthes bidentata, Acmella oleraceo,Actaea racemosa, Actinidia chinensis, Actinidia deliciosa, Adansoniadigitata, Adiantum capillus-veneris, Aesculus hippocastanum, Afromomummelegueta, Agathosma betulina, Agathosma crenulata, Agathosmaserratifolia, Agrimonia eupatoria, Ajuga reptans, Albizia julibrissin,Alchemilia vulgaris, Micro petiolata, Allium ampeloprasum, Allium cepa,Allium sativum, Allium schoenoprasum, Allium ursinum, Allium glutinosa,Aloe ferox, Aloe vera, Aloysia citriodora, Alpinia galanga, Alpiniahainonensis, Alpinia officinarum, Alpinia oxyphyllo, Althaeaofficinalis, Ammi visnaga, Amorphophallus konjac, Ananas comosus,Andographis paniculata, Anemarrhena asphodeloides, Anethum graveolens,Angelica archangelica, Angelica dahurica, Angelica pubescens, Angelicasinensis, Antennaria diocia, Anthriscus cerefolium, Anthyllisvulneraria, Aphanizomenon flos-aquae Ralfs, Apium graveolens, Arachishypogaea, Aralia data, Arctium lappa, Arctium minus, Argania spinosa,Armorica rustanica, Artemisia dracunculus, Artemesia vulgaris,Ascophyllum nodosum, Aspalathus linearis, Asparagus officinal's,Astragalus membranaceus, Atractylodes lancea, Atractylodes macrocephala,Auracaria columnaris, Avena staiva, Ayahuasca, Baccharis genistelloides,Bacopa monnierri, Ballota nigra, Bambusa bambos, Bellis perennis,Berberis vulgaris, Beta vulgaris, Betula alleghaniensis, Betula pendula,Betula pubescens, Bixa orellana, Borago officnalis, Boswellia serrata,Brassica napus, Brassica nigra, Brassica oleracea, Brassica rapa,Bupleurum chinense, Calendula officinalis, Calluna vulgaris, Camelliasinsensis, Capseila bursa-pastoris, Capsicum annuum, Carex arenaria,Carica papaya, Carlina acoulis, Carphephorus odoratissmus, Carpinusbetulus, Carthamus tinctorius, Carum carvi, Cassia fistula, Castaneasativa, Centaurea centaurium, Centaurea cyanus, Centourium erythraeo,Centella asiatica, Cerasus vulgaris, Ceratonia silliqua, Chaenomelumnobile, Chlorella vulgaris, Chondrus crispus, Chrysanthellum indicum,Cichorium intybus, Cinchona officinalis, cinchona pubescens, Cinnamomumcamphora, Cinnamomum cassia, Cinnamomum verum, Cistanche salsa, Cistusincanus, Citrus aurantium, Citrus limon, Citrus maxima, Citrus medica,Citrus myrtifolia, Citrus reticulata blanco, Citrus sinsensis, Citrusparadisi, Clinopodium vulgare, Cnicus benedictus, Cochleariaofficinalis, Cocos nucifera, Codonopsis pilosula, Coffea canephora, Coixlacryma-jobi var. mayyuen Stapf, Cola acuminata, Cola ballayi cornu,Cola nitida, Combretum micranthum, Commiphora mukul, Conyza canadensis,Coriandrum sativum, Cornus officinalis, Corylus avellana, Corymbiacitriodora, Crataegus laevigata, Craetegus monogyna, Crithmum maritimum,Crocus sativus, Cucumis melo, Cucurbita pepo, Cuminum cyminum, Cupressussempervirens, Cuscuta chinensis, Cyamopsis tetragonoloba, Cyathulaofficinalis, Cyclanthera pedata, Cydonia oblonga, Cymbopogon martini,Cymbopogon nardus, Cymbopogon winterianus, Cynara cardunculus, Cyperusrotundus, Daucus carota, Dendranthema grandiflorum, Desmodiumadscendens, Dimocarpus longan, Dioscorea oppostifolia, Dioscoreavillosa, Diospyros kaki Thunb., Dunaliella saliena, Echinaceaaugustifolia, Echinacea pallida, Echinacea purpurea, Elaegnusrhamnoides, Alettaria cardamomum, Eleutherococcus senticosus, Elymusrepens, Epiobium augustifolium, Epilobium parviflorum, Equisetumarvense, Erica cinerea, Erica tetralix, Eriobotrya japonica, Eriodictyoncalifornicum, Erodium cicutarium, Eryngium campestre, Eschscholziacalifornica, Eucalyptus dives Schauer, Eucalyptus globulus, Eucalyptusradiata, Eucalyptus smithii F. Muell, Eucommia ulmoides, Eugeniauniflora, Eugenia jambolana, Euphrasia stricta D. Wolff, Euterpeoleracea, Fagopyrum esculentum Moench, Follopia japonica, Ferulaassa-foetida, Ficus carica, Filipendula ulmaria, Foeniculum vulgareMill., Forsythia suspensa, Fragaria dodonei Ard., Frangula purshianaCooper, Fraxinus excelsior, Fraxinus ortus, Fucus serratus, Fucusvesiculosus, Fumaria officinalis, Galeopsis segetum Neck., Galiumodotarum, Galium verum, Gardenia jasminoides J. Ellis, Gastrodia elataBlume, Gelidium corneum J. V. Lamouroux, Gentiana lutea, Geraniumrobertianum, Geum urbanum, Ginkgo biloba, Glycine max, Glycyrrhizaglabra, Glycyrrhiza uralensis, Gracilaria gracilis, Grindelia camporumGreene, Grindelia robusta Nutt., Grindelia squarrosa Dunal, Gymnemasylvestris, Haematococcus pluvialis, Hamamemis virginiana, Harpagophytumprocumbens, Harpagophytum zeyheri Decne., Hedeoma pluegioides Pers.,Helianthus annuus, Helienthus tuberosus, Helichrysum arenarium,Helichrysurn stoechas, Herniara glabra, Hibiscus sabdariffa, Hieraciumpilosella, Himanthalia elongata, Hordeum vulgare, Houttuynia cordataThunb., Huperzio serrata, Hyssopus officinalis, Ilex paraguariensis A.St.-Hill, Illicum verum, Impatients balsamina, Inula britannica, Inulahelenium, Jasminum grandiflorum, Jasmium officinale, Juniperus communis,Justicia adhatoda, Kavalama urens, Krameria lappacea, Lagerstroemiaspeciosa, Laminaria digitata, Laminaria hyperborea, Lamium album, Larixdeciduo, Larix occidentalis, Laurus nobilis, Lavandula augustofolio,Lavandula latifolia, Ledum palustre, Leonurus cardiaca, Lepidium meyeniiWalp., Lepidium sativum, Lespedeza capitata, Levisticum officinale,Lindera aggregata, Linus usitatissimum, Liquidambar styraciflua, Lotuscorniculatus, Lyciutn barbarum, Lycium chinense, Lycopersiconesculentum, Lycopodium clavatum, Lycopus europaeus, Lythrum salicaria,Macadamia ternifolia F. muell, Macrocystis pyrifera, Magnoliaofficinalis, Malpighia glabra, Malus pumila, Malus domestica, Malussylvestris, Malva sylvestris, Mangifera indica, Maranta arundinacea,Marrubium vulgare, Marsdenia cundurango, Marsdenia sylvestris,Mastocarpus stellatus, Matricaria chamomilla, Medicago sativa, Melaleucaalternifolia, Melaleuca cajuputi Powell, Melaleuca leucadendra,Melaleuca quinquenrvia, Melaleuca viridiflora, Melilotus altissimusThuill., Melillotus officinalis, Mentha arvensis, Mentha× piperita,Menyanthes trifoliata, Mesembryanthemum crystallinum, Monarda didyma,Morinda citrifolia, Morinda officinalis, Morus alba, Morus nigra,Murraya koenigii, Musa× paradisiaca, Myrciaria dubia, Myristica flagransHoutt., Myroxylon balsamum, Myrtus communis, Nardostachys jatamansi,Nasturtium officinale R. Br., Nelumbo nucifera Gaertn., Nepeta cataria,Nepeta tenuifolia Benth., Nigella sativa, Ocimum basilicum, Oenotherabiennis, Ononis spinosa, ophiopogon japonicus, Opuntia ficus-indica,Origanum compactum Benth., Origanum majorana, Origanum vulgare,Orthosiphon aristatus, Oryza sativa, Paeonia lactiflora, Paeonia×suffruticosa Andrews, Palmaria palmata, Panax ginseng, Panaxquinquefolius, Panicum miliacium, Papaver rhoeas, Parietariaofficinalis, Passiflora edulis Sims, Pastinaca sativa, Paullinia cupanaKunth, Pelargonium graveolens, Perilla frutescens, Persea americana,Persicaria bistorta, Persicaria maculosa Gray, Petroselinum crispum,Peucadanum ostruthium, Peumus boldus Molina, Phaseolus vulgaris,Phellodendron amurense, Photinia meloncorpa, Phyllanthus emblica,Physalis alkekengi, Phymatolithon calcareum, Picea abies, Pimento dioca,Pimento racemosa, Pimpinella anisum, Pimpinello major, Pimpinellasaxtraga, Pinus muga Turra, Pinus pinaster Aiton, Pinus sylvestris,Pistacia lentiscus, Plantago arenaria, Plantago lanceoiata, Plantagomajor, Plantago ovata, Platycodon grandiflorus, Plectranthus barbatusAndrews, Pogostemom cablin, Polygala senega, Polygala sibirica, Polygalatenuifolia Wild., Polygonum aviculare, Populus nigra, Populus tremula,Populus tremuloides, Porphyra umbilicalis, Portulaca oleracea,Potentilla erecta, Primula veris, Prunella vulgaris, Prunus africana,Prunus armeniaca, Ribes nigrum, Ribes uva-crispa, Rosa canina, Rosagallica, Rosa moschata, Rosa rubiginosa, Rosmarinus officinalis, Rubuscaesius, Rubus fruticosus, Rubus idaeus, Rumex actetosa, Rumexacetosella, Rumex crispus, Rumex patienta, Ruscus aculeatus, Sachharinajaponica, Saccharina latissima, Salix alba, Salix fragilis, Salixpentandra, Salix purpurea, Salvia officinalis L., Salvia officinalissubsp. lavandulfaiia Gams, Salvia sclarea, Sambucus nigra, Sanguisorbaofficinalis, Sanicula elata Buch.-Ham. Ex D. Don, Santalum album,Santolina chamaecyparissus, Saposhnikovia divaricata, Sargassumfusiforme, Satureja hortensis, Satureja montana, Saussurea costus,Scrophularia ningpoensis Helmsl., Scutellaria baicalensis Georgi, Secalecereale, Sedum acre, Sedum roseum, Senna alexandrina Mill., Sennaobustifolia, Smilax cordifolia Humb. & Bonpl., Smilax glabra Roxb.,Smilax officinalis Kunth, Smilax purhampuy Ruiz, Smilax purhampuy Ruiz,Smilax regelli Killip and C. V. Morton, Smilax vanillidora Apt, Solanummelongena, Solanum tuberosum, Solidago virgaurea, Sorbus aucuparia,Spatholobus suberctus Dunn., Spinacia oleracea, Spirulina major Kützing,Spirulina maxima Geitler, Spirulina platensis Geitler, Stavhysofficinalis, Stemmacantha carthamoides Dittrich, Styphoiobium japonicum,Syzgium aromaticum, Tagetes erecta, Tamarindus indica, Tanacetumparthemium, Terminalia chebula Retz., Theobroma cacao, Thymussaturejoides Coss., Thymus serpyllum, Thymus vulgaris, Thymus zygis,Tilia cordata Mill., Tilia platyphyllos Scop., Tilia tomentosa Moench,Tilia euopoea, Tribulus terrestris, Trichosanthes kirilowii Maxim.,Trifolium arvense, Trifolium campestre Schreb., Trifolium pratense,Trifolium repens, Trigonella caerulea, Trigonella foenum-graecum,Tricitum aestivum, Tricitum durum Desf., Tricitum spelta L., Tricitumturgidum, Tropaeolum majus, Turnera diffusa Willd., Ulmus glabra Huds.,Ulmus glabra Huds., Ulmus pumila, Ulmus rubra Muhl., Ulva lactuca,Uncaria gambir Roxb., Uncaria rhynchophylla Miq., Uncoria tomentosa DC.,Undaria pinnatifida Suringar, Urtica dioca, Urtica urens, Vacciniummacrocarpon, Vaccinium oxycoccos, Vaccinium vitis-idae, Valerianajatamansi Jones, Valeriana officinalis, Vanilla planifolia Jacks,Verbascum densiflorum Bertol., Verbascum thapsus, Verbena officinalis,Veronica officinalis, Viburnum opulus, Vigna angularis Ohwi & H. Ohashi,Vinca major, Vinca minor, Viola palustris, Viola tricolor, Vitexagnus-castus, Vitex trifoiia, Vitis vinifera, Zea mays, Zingiberofficinale Roscoe, Ziziphus jujuba Mill.

The compositions according to the invention may be in any form,especially in the form of powder, gel, emulsion or in liquid form, inparticular in the form of tablets, wafer capsules, gel capsules, sticks,sachets, vials, droppers or in injectable form.

The compositions according to the invention may be used as nutritionproducts or health products in particular as medicaments.

The term “nutrition product” means any product having a nutritionaland/or physiological effect, this especially comprising foodsupplements, foods, dietetic products, etc. These products may inparticular be administered via the oral, gastric or venous route.

The term “health product” means any product which has a beneficialeffect on health, in prevention or treatment, whether this effect isphysiological or pharmacological, especially medicaments, pharmaceuticalproducts. These products may in particular be administered via the oral,gastric, venous or cutaneous route.

The compositions according to the invention may be used for preventingand/or combating carbohydrate and/or fat metabolism disorders in Humanor animals.

They are particularly suitable for preventing and/or combating type 2diabetes in Human or animals. Specifically, they make it possible toprevent the establishment of chronic hyperglycemia, to decrease fastingglycemia and glycated hemoglobin, circulating and hepatic triglycerides,body mass and fat mass, to increase HDL cholesterol (“good cholesterol”)and to improve the tolerance to ingested carbohydrates and insulinsensitivity. They may be used preventively in the treatment of type 2diabetes and as a first line treatment, during the establishment ofHDMs, thus making it possible to defer the implementation of the usualoral antidiabetic molecules. They are also particularly suitable fortreating type 2 diabetes and its complications, non-alcoholicsteatohepatitis (NASH) especially, alone or in combination with otherpharmacological treatments.

The compositions according to the invention may also be used forpreventing and/or combating type 1 diabetes and/or non-alcoholic fattyliver diseases, in particular non-alcoholic steatohepatitis (NASH),and/or cardiovascular pathologies, in particular coronary cardiopathies,cerebrovascular diseases, peripheral arteriopathies, deep veinthrombosis, and/or pathologies associated with insulin resistance, forexample Alzheimer's disease (Bedse G et al. Front Neurosci 2015; 9:204).

For such uses, the compositions according to the invention may be usedin combination with at least one antidiabetic therapeutic agent chosenfrom biguanides including metformin, dipeptidyl peptidase-IV (DPP-IV)inhibitors, glucagon-like peptide-1 (GLP-1) analogs, thiazolidinediones(TZDs), sulfonylureas, rapid and slow insulins, glycosidase inhibitors(acarbose, miglitol, voglibose), sodium glucose co-transporter-2 (SGLT2)inhibitors, molecules of the fibranor family such as elafibranor, ormolecules targeting nuclear receptors and especially the ROR (α, β, γ)receptors and Rev-Erb (α, β) receptors.

The compositions according to the invention may also be used for actingon other cardiovascular risk factors or metabolic syndrome.

In particular, the compositions according to the invention may be usedfor preventing and/or combating dyslipidemia. They especially have ahypocholesterolemiant effect and make it possible to reduce the totallevel of cholesterol, the level of LDL cholesterol, the circulatingtriglycerides and the hepatic triglycerides. They also have inhibitoryactivity on HMG-CoA reductase.

For such uses, the compositions according to the invention may be usedin combination with a hypolipemiant therapeutic agent chosen from:statins, fibrates, nicotinic acid, ion-exchange resins, cholesterolabsorption inhibitors, omega-3 polyunsaturated fatty acids, tiadenol,and FXR (Farnesoid X Receptor) nuclear receptor agonists.

Finally, the compositions according to the invention may be usedspecifically for preventing or combating obesity and excess weightand/or metabolic syndrome and/or pathological arterial tension problems.

The invention is now illustrated by examples of extracts and ofcompositions, and also by results of tests demonstrating the efficacy ofthe compositions according to the invention, these examples and testsnot being limiting.

I. EXAMPLES Example 1 Example of Dry Extract of Chrysanthellum indicum

The aerial parts of the fresh or dry plant are subjected to mechanicalgrinding until a coarse powder is obtained. This powder is thensubjected to a maceration step for 10 to 24 hours at room temperature ina 70/10 water/ethanol mixture, and the mix obtained is then subjected tocontinuous leaching at 50° C. in a percolator with a 70/10 water/ethanolmixture, the plant/extract ratio being 3/1. The extract obtained is thensubjected to liquid/liquid washes using a nonpolar organic solvent suchas dichloromethane or trichloromethane. After concentrating byevaporation at low pressure at 35° C., a liquid is obtained, which islyophilized for 24 hours to give a beige-colored powder that is solublein a water/alcohol mixture. This powder (dry extract) may be useddirectly or may be mixed in a suitable solvent before use.

Example 2 Example of Dry Extract of Vaccinium myrtillus

Blueberry in powder form obtained from Vaccinium myrtillus fruit issubjected to a maceration step for 10 to 24 hours at room temperature ina 30/50 water/ethanol mixture and the mix obtained is then subjected tocontinuous leaching at 50° C. in a percolator with a 30/50 water/ethanolmixture, the plant/extract ratio being 10/1. The extract obtained isthen subjected to liquid/liquid washes using a nonpolar organic solventsuch as dichloromethane or trichloromethane. After concentrating byevaporation at low pressure at 35° C., a liquid is obtained, which islyophilized for 24 hours to give a violet-colored powder which issoluble in a water/alcohol mixture.

Example 3 Example of Dry Extract of Cynara scolymus

Artichoke in powder form obtained from leaves of Cynara scolymus issubjected to a step of maceration for 10 to 24 hours at room temperaturein water, and the mix obtained is then subjected to continuous leachingat 50° C. in a percolator with water, the plant/extract ratio being 2/1.The extract obtained is then subjected to liquid/liquid washes using anonpolar organic solvent such as dichloromethane or trichloromethane.After concentrating by evaporation at low pressure at 35° C., a liquidis obtained which is lyophilized for 24 hours to give a beige-coloredpowder that is soluble in water.

Example 4 Example of Dry Extract of Olea europaea

Air-dried whole olive tree leaves are ground at −80° C. using a knifemill to obtain a fine and uniform powder. The powder obtained is thensubjected to a maceration step for 10 to 24 hours in a 70/30water/ethanol mixture. The step is performed in a closed system with anitrogen sparge at room temperature, or under a microwave power of 800watts or under an ultrasound frequency of 20 kHz for 2× 3 min. The mixobtained is then subjected to continuous leaching at 50° C. in apercolator with a 70/30 water/ethanol mixture, the plant/extract ratiobeing 10/1. The extract obtained is then subjected to liquid/liquidwashes using a nonpolar organic solvent such as dichloromethane ortrichloromethane. After concentrating by evaporation at low pressure at35° C., a liquid is obtained, which is lyophilized for 24 hours to givea green-colored powder in a water/alcohol mixture.

Example 5 Examples of Single Extract

Chrysanthellum in powder form obtained from the aerial parts ofChrysanthellum indicum, and artichoke in powder form obtained fromCynara scolymus leaves, and blueberry in powder form obtained fromVaccinium myrtillus fruit, and Piper nigrum fruit powder, and olive treein powder form obtained from Oleo europaea leaves are subjected to astep of maceration for 10 to 24 hours at room temperature in a 40/60water/ethanol mixture, and the mix obtained is then subjected tocontinuous leaching at 50° C. in a percolator with a 40/60 water/ethanolmixture, the mixture of plants/single extract ratio being from 4 to 6/1.The extract obtained is then subjected to liquid/liquid washes using anonpolar organic solvent such as dichloromethane or trichloromethane.After concentrating by evaporation at low pressure at 35° C., a liquidis obtained, which is lyophilized for 24 hours to give a violet-coloredpowder that is soluble in a water/alcohol mixture.

Example 6 Example of a Composition According to the Invention in theForm of Tablets, Comprising Four Plant Extracts

The composition of example 6 is in the form of tablets that may beadministered orally. It comprises, expressed as weight percentages,relative to the total weight of the composition, 30.1% of dry extract ofaerial parts of Chrysanthellum indicum, 30.1% of dry extract of Cynarascolymus leaves, 3.0% of dry extract of Vaccinium myrtillus fruit and0.3% of dry extract of Piper nigrum fruit. It also comprises excipients,in particular microcrystalline cellulose and magnesium stearate.

The composition for three tablets is indicated in Table 1 below.

TABLE 1 Example of composition in tablet form For List of ingredientsthree tablets Dry extract of aerial parts of Chrysanthellum indicum 600mg Dry extract of Cynara scolymus leaves 600 mg Dry extract of Vacciniummyrtillus fruit 60 mg Dry extract of Piper nigrum fruit 6 mgMicrocrystalline cellulose 700 mg Magnesium stearate 26 mg

Example 7 Example of a Composition According to the Invention in theForm of Gel Capsules, Comprising Five Plant Extracts

The composition of example 7 is in the form of gel capsules that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 37.0% of dry extract ofaerial parts of Chrysanthellum indicum, 37.0% of dry extract of Cynarascolymus leaves, 3.7% of dry extract of Vaccinium myrtillus fruit,0.004% of dry extract of Piper nigrum fruit, and 22.2% of dry extract ofOleo europaea leaves.

The composition for three gel capsules is indicated in Table 2 below.

TABLE 2 Example of composition in the form of gel capsules For threeList of ingredients gel capsules Dry extract of aerial parts ofChrysanthellum indicum 200 mg Dry extract of Cynara scolymus leaves 200mg Dry extract of Vaccinium myrtillus fruit 20 mg Dry extract of Pipernigrum fruit 0.02 mg Dry extract of Olea europaea leaves 120 mg

Example 8 Example of a Composition According to the Invention in theForm of Tablets, Comprising Five Plant Extracts

The composition of example 8 is in the form of tablets that may beadministered orally. It comprises, as weight percentages relative to thetotal weight of the composition, 22.0% of dry extract of the aerialparts of Chrysanthellum indicum, 22.0% of dry extract of Cynara scolymusleaves, 2.2% of dry extract of Vaccinium myrtillus fruit, 13.2% of dryextract of Oleo europaea leaves, and 0.2% of dry extract of Piper nigrumfruit. The composition also comprises, in addition to the mixture, zinc,vitamins B9, PP, B5, H, B12, D, B6, B2, B2 and chromium. It alsocomprises excipients, in particular dicalcium phosphate,microcrystalline cellulose and magnesium stearate.

The composition for one tablet is indicated in Table 3 below.

TABLE 3 Example of composition in the form of tablets Referencenutritional In mg for value for List of ingredients one tablet onetablet Dry extract of aerial parts of 220.0 — Chrysanthellum indicum Dryextract of Cynara scolymus leaves 220.0 — Dry extract of Vacciniummyrtillus fruit 22.0 — Dry extract of Olea europaea leaves 132.0 — Dryextract of Piper nigrum fruit 2.0 — Dicalcium phosphate 198.0 —Microcrystalline cellulose 153.44 — Magnesium stearate 20.0 — Zincbisglycinate 12.99  33% Vitamin B9 - folic acid 7.30  33% Vitamin PP -nicotinamide 5.30 33.1% Vitamin B5 - calcium pantothenate 2.24 33.3Vitamin H - biotin 1.66 33.2% Vitamin B12 0.83 33.2% Vitamin D3 0.64 32% Vitamin B6 - pyridoxine hydrochloride 0.56 32.9% Vitamin B1 -thiamine hydrochloride 0.48 32.7% Vitamin B2 - riboflavin 0.45 32.1%Chromium picolinate 0.11 32.5%

Example 9 Example of a Composition According to the Invention in theForm of Tablets, Comprising Four Plant Extracts

The composition of example 9 is in the form of tablets that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 23.9% of dry extract ofChrysanthellum indicum whole plants, 23.9% of dry extract of Cynarascolymus leaves, 23.9% of dry extract of Vaccinium myrtillus fruit and0.2% of dry extract of Piper nigrum fruit. The composition alsocomprises vitamin B1, vitamin B3, pantothenic acid (vitamin B5), zincand chromium. As excipients, it comprises microcrystalline cellulose anddicalcium phosphate. As coating agents, it comprises talc, sugar,shellac, povidone and beeswax.

The composition for three tablets is indicated in Table 4 below.

TABLE 4 Example of composition in tablet form Reference nutritional Forvalue for List of ingredients three tablets three tablets Dry extract ofVaccinium myrtillus 600 mg — fruit Dry extract of Chrysanthellum indicum600 mg — whole plants Dry extract of Cynara scolymus leaves 600 mg — Dryextract of Piper nigrum fruit 5 mg — Vitamin B1 1.1 mg 100% Vitamin B3(inositol hexanicotinate) 16 mg 100% (nicotinic acid) Pantothenic acid(vitamin B5) 6 mg 100% Zinc (zinc bisglycinate) 5 mg  50% Chromium(chromium picolinate) 40 μg 100% Microcrystalline cellulose 600 mg —Dicalcium phosphate 75 mg —

Example 10 Example of a Composition According to the Invention in theForm of a Powder to be Reconstituted in Water, Conditioned in the Formof Sticks, Comprising Four Plant Extracts

The composition of example 10 is in the form of a powder to bereconstituted in water, conditioned in the form of sticks that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 36.3% of dry extract ofChrysanthellum indicum whole plants, 24.2% of dry extract of Cynarascolymus roots, 36.3% of dry extract of Vaccinium myrtillus fruit and2.4% of dry extract of Piper longum fruit. The composition alsocomprises vitamin B1, vitamin B3, pantothenic acid (vitamin B5), zincand chromium. As flavor, it comprises a natural blueberry flavor. Asacidifying agent, it comprises malic acid. As antiagglomerating agent,it comprises silicon dioxide, as thickener, xanthan gum, and asstabilizer, calcium phosphate.

Its composition is indicated in Table 5 below.

TABLE 5 Example of composition in the form of powder to be reconstitutedin water and conditioned in the form of sticks Reference nutritional Forvalue for List of ingredients three sticks three sticks Dry extract ofVaccinium myrtillus 1500 mg — fruit Dry extract of Chrysanthellumindicum 1500 mg — whole plants Dry extract of Cynara scolymus roots 1000mg — Dry extract of Piper longum fruit 100 mg — Vitamin B1 1.1 mg 100%Vitamin B3 (inositol hexanicotinate) 16 mg 100% (nicotinic acid)Pantothenic acid (vitamin B5) 6 mg 100% Zinc (zinc bisglycinate) 5 mg 50% Chromium (chromium picolinate) 40 μg 100%

Example 11 Example of a Composition According to the Invention in theForm of a Powder to be Reconstituted in Water, Conditioned in the Formof Sticks

The composition of example 11 is in the form of a powder to bereconstituted in water, conditioned in the form of sticks that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 37.0% of dry extract ofaerial parts of Chrysanthellum indicum, 37.0% of dry extract of Cynarascolymus leaves, 3.7% of dry extract of Vaccinium myrtillus fruit, 0.2%of dry extract of Piper nigrum fruit, and 22.2% of dry extract of Oleaeuropaea leaves. The composition also comprises vitamin B12 andchromium. As flavor, it comprises a natural strawberry flavor. Asantiagglomerating agent, it comprises silicon dioxide. This compositiondoes not comprise any antiagglomerating agent and thickener.

The composition of such a product is indicated in Table 6 below.

TABLE 6 Example of composition in the form of powder to be reconstitutedin water and conditioned in the form of sticks Reference nutritional Forvalue for List of ingredients three sticks three sticks Dry extract ofVaccinium myrtillus 120 mg — fruit Dry extract of aerial parts of 1200mg — Chrysanthellum indicum Dry extract of Cynara scolymus leaves 1200mg — Dry extract of Piper nigrum fruit 6 mg Dry extract of Olea europaealeaves 720 mg — Vitamin B3 (inositol hexanicotinate) 2.5 μg 100%Chromium (chromium picolinate) 20 μg  50%

Example 12 Example of a Composition According to the Invention in theForm of Gel Capsules

The composition of example 12 is in the form of gel capsules that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 31.6% of dry extract ofthe aerial parts of Chrysanthellum indicum, 47.4% of dry extract ofCynara scolymus roots, 15.8% of dry extract of Vaccinium myrtillus fruitand 2.6% of piperine. The composition also comprises vitamin B3 andzinc. As emulsifier, It comprises soybean lecithin derived from non-GMOproduction, as thickeners, colloidal silica and fatty acid mono- anddiglycerides. The capsule is a fish gelatin, with glycerol and acolorant, red iron oxide.

The composition of such a product is indicated in Table 7 below.

TABLE 7 Example of composition in the form of gel capsules Referencenutritional For two value for two List of ingredients gel capsules gelcapsules Dry extract of Vaccinium myrtillus 100 mg — fruit Dry extractof the aerial parts of 200 mg — Chrysanthellum indicum Dry extract ofCynara scolymus roots 300 mg — Piperine  15 mg — Vitamin B3  8 mg  50%(nicotinic acid) Zinc (zinc gluconate)  10 mg 100%

Example 13 Example of a Composition According to the Invention in theForm of Gel Capsules

The composition of example 13 is in the form of gel capsules that may beadministered orally. It comprises, expressed as weight percentagesrelative to the total weight of the composition, 10.4% of dry extract ofChrysanthellum indicum whole plants, 20.7% of dry extract of Cynarascolymus leaves, 62.1% of dry extract of Vaccinium myrtillus leaves and2.6% of piperine. As filler, it comprises corn starch. Asantiagglomerating agents, it comprises silicon dioxide and magnesiumstearate. The gel capsule is of plant origin. The composition of such aproduct is indicated in Table 8 below.

TABLE 8 Example of composition in the form of gel capsules Referencenutritional For one value for one List of ingredients gel capsule gelcapsule Dry extract of Vaccinium myrtillus 300 mg — leaves Dry extractof Chrysanthellum indicum  50 mg — whole plants Dry extract of Cynarascolymus leaves 100 mg — Piperine  15 mg —

Example 14 Example of a Composition According to the Invention in theForm of Gel Capsules Comprising a Single Extract

The composition of example 14 is in the form of gel capsules that may beadministered orally. It comprises a single aqueous-alcoholic extract ofa powder mixture obtained from the aerial parts of Chrysanthellumindicum, from Cynara scolymus leaves, from Vaccinium myrtillus fruit,from Piper nigrum fruit and from Oleo europaea leaves. The ratio betweenthe three plants is 1/1/0.1/0.0001/0.6.

-   -   The single extract of the mixture of plants may be obtained via        any suitable process, for example via a process comprising the        following steps:    -   solid/liquid extraction    -   separation/pressing    -   filtration    -   evaporation    -   drying    -   optionally incorporation of additives    -   homogenization    -   conditioning.

Example 15 Example of a Composition According to the Invention in theForm of Tablets Comprising Synthetic Molecules or Molecules Obtainedfrom Plant Raw Materials

The composition of example 15 is in the form of film-coated tablets thatmay be administered orally. It comprises as active substance for onetablet: 50 mg of apigenin 7-O-glucuronide, 50 mg of dicaffeoylquinicacid, 100 mg of monocaffeoylquinic acid, 10 mg of piperine and 250 mg ofoleuropein. The other components used as excipients are: pregelatinizedstarch, sodium carboxymethyl starch (type A), stearic acid, povidoneK90, anhydrous colloidal silica.

The active substances may be synthetic or derived from plant rawmaterials or derived from plant extracts by purification byhigh-performance liquid-phase chromatography.

II. IN VIVO EVALUATION OF THE EFFICACY OF THE COMPOSITION

in vivo experiments on mice were performed to demonstrate the effects ofthe compositions according to the invention, in particular on fastingglycemia, glycated hemoglobin, carbohydrate tolerance, insulinsensitivity, body mass and fat mass, and on the circulating and hepaticfats. Similarly, molecular biology evaluations were performed. Finally,the compositions were compared with reference pharmacological treatmentsalready on the market or undergoing development.

The experiments were performed on db/db mice. db/db mice have a mutationof the leptin receptors inducing a cell signaling dysfunction of thelatter. The leptin receptors are highly expressed in the hypothalamus.Mice having a mutation of these receptors cannot efficiently regulatetheir energy stores. This results in high insulinemia from the firstdays of life (10-14 days), and obesity from 3 to 4 weeks with anincrease in glycemia. These mice are insulin resistant,hypertriglyceridemic and glucose-intolerant. They constitute a pertinentand predictive model especially for studying insulin sensitivity,triglyceridemia, type 2 diabetes and one of its complications,non-alcoholic steatohepatitis (NASH) (Aileen J F King Br J Pharmacol2012; 166(3):877-894; Sanches S C et al. Biomed Res Int 2015).

II.1 TESTS A

The experimental time was nine weeks with a “run-in” of one weekfollowed by eight weeks of supplementation with the plant extracts and acomposition X. The male mice were ten weeks old at the start of thetreatment.

Nine compositions X were tested. These compositions were directlyincorporated into the rodents' feed, which makes it possible to ensureits “multi-target” efficacy and its large-scale use, since intravenousor intraperitoneal injections are limited to a small number of people,given their mode of administration. This also avoids dailyforce-feeding, which alters various physiological processes.

The compositions tested were as follows:

-   -   C1: Chrysanthellum indicum (whole plant)+piperine (1% and 0.1%        of the feed, respectively);    -   C2: Cynara scolymus (leaves)+piperine (1% and 0.1% of the feed,        respectively);    -   C3: Vaccinium myrtillus (fruits)+piperine (1% and 0.1% of the        feed, respectively);    -   C4: Chrysanthellum indicum (whole plant)+Cynara scolymus        (leaves)+Vaccinium myrtillus (fruits)+piperine (1%, 1%, 1% and        0.1% of the feed, respectively);    -   C5: piperine (0.1% of the feed, respectively);    -   C6: Chrysanthellum indicum (whole plant)+Cynara scolymus        (leaves)+Vaccinium myrtillus (fruits) (1%, 1% and 1% of the        feed, respectively);    -   C7: Chrysanthellum indicum (whole plant)+Cynara scolymus        (leaves)+Vaccinium myrtillus (fruits)+piperine (1%, 1%, 1%,        0.001% of the feed, respectively);    -   C8: Oleo europaea (leaves) (0.6% of the feed, respectively);    -   C9: Chrysanthellum indicum (whole plant)+Cynara scolymus        (leaves)+Vaccinium myrtillus (fruits)+piperine+Olea europaea        (leaves) (1%, 1%, 1%, 0.001%, 0.6% of the feed, respectively).

The term “piperine” means either synthetic piperine or a standardizedPiper extract containing 95% piperine. For the other plants, they aredry extracts obtained from plant raw materials.

The experiments took place in several steps. Consequently several “adlibitum control” groups were made. The results obtained from thesegroups were pooled. Compositions C1, C2, C3, C4, C5, C8 and C9 induced asimilar decrease in feed intake. Consequently, a “Per Fed control”group, i.e. a group consuming the same daily amount of food as groupsC1, C2, C3, C4, C5, C8 and C9, was made so as to be able to compare theresults for an equivalent feed intake.

The experimental evaluations focused especially on:

-   -   Measurement of the body mass;    -   Measurement of the fasting glycemia;    -   The change in glycemia during an oral test of carbohydrate        tolerance. After force-feeding with starch (3 g/kg) in the        fasted state, the change in glycemia in response to starch was        measured in the tail by biopsy just before the force-feeding        (t0) and then after 30, 60, 90 and 120 minutes. The area under        the curve (AUC) was calculated. An increase in the AUC reflects        a carbohydrate intolerance, a decrease reflects an improvement        in carbohydrate tolerance.

For compositions C7 to C9, an insulin sensitivity test was alsoperformed. This test consisted of an intraperitoneal injection ofinsulin (2 U/kg) in the fasted state. The change in glycemia in responseto the insulin injection was measured in the tail by biopsy just beforethe injection (t0) and then after 30, 60, 90 and 120 minutes. The areaunder the curve (AUC) was calculated. A decrease in the AUC reflects abetter response to the insulin injection and thus an improvement in theinsulin sensitivity. Conversely, an increase in the AUC reflects poorerinsulin sensitivity and thus insulin resistance.

The evaluations presented were performed just before the supplementation(t=0) and at the end of supplementation (t=8 weeks).

The results obtained are presented in tables:

-   -   Table 9 for the effect on the insulin sensitivity after eight        weeks of treatment,    -   Table 10 for the effect on the fasting glycemia after eight        weeks of treatment,    -   Table 11 for the effect on the carbohydrate tolerance after        eight weeks of treatment,    -   Table 12 for the effect on the body mass after eight weeks of        treatment.

Insulin Sensitivity

TABLE 9 Effect of the compositions on the insulin sensitivity aftereight weeks of treatment AUC (week 8 − week 0, in mg × min/dL)Compositions (mean ± SEM) ad libitum control 19 186 ± 3460   C7  2082 ±2082 ** C8 8970 ± 2959  C9 −6520 ± 4685 ** Mean values ± SEM. ad libitumcontrol, n = 20; C7, n = 11; C8, n = 10; C9, n = 9. Composition versuscontrol, Student's t test for unpaired data, ** p < 0.01.

The results presented in Table 9 are also illustrated in FIG. 1. Theseresults show a very significant and large effect of compositions C7 andC9 on the insulin sensitivity. The improvement in insulin sensitivitywith compositions C7 and C9 according to the invention is particularlyadvantageous in prevention and in treatment of type 2 diabetes andcomplications thereof. Specifically, insulin resistance is one of themajor mechanisms of progression of type 2 diabetes (Samuel V T et al.Cell 2012; 148:852-71). Moreover, by acting on this parameter,compositions C7 and C9 according to the invention are particularlysuitable in other pathologies in which one of the major causes isinsulin resistance. This is particularly the case for one of thecomplications of type 2 diabetes, non-alcoholic steatohepatitis (NASH;Samuel V T et al. Cell 2012; 148:852-71), and Alzheimer's disease (BedseG et al. Front Neurosci 2015; 9:204).

Fasting glycemia

TABLE 10 Effect of the compositions on the fasting glycemia after eightweeks of treatment Fasting glycemia Compositions (in mg/dL) ad libitumcontrol 534 ± 10 Per Fed control 518.2 ± 38  C1 410.4 ± 33 * C2 448 ± 34C3  384 ± 40 * C4   335 ± 38 ** C5 449 ± 11 C6 536 ± 15 C7  571 ± 14 *C8 400 ± 61 C9   220 ± 78 ** Mean values ± SEM. ad libitum control, n =32; Per Fed control, n = 10; C1, n = 9; C2, n = 9; C3, n = 9; C4, n = 8;C5, n = 9; C6, n = 14; C7, n = 12; C8, n = 12; C9, n = 9. Compositionversus control, Student's t test for unpaired data, * p < 0.05, ** p <0.01.

Compositions C1, C2, C3, C4, C5, C8 and C9 brought about a decrease infeed intake. Consequently, a Per Fed group consuming the same amount offood was made. The results show that the calorie restriction imposed onthe animals had no effect on the fasting glycemia (comparison of adlibitum control versus Per Fed control). Compositions C1, C3, C4 and C9induced a significant decrease in fasting glycemia, the most pronouncedeffect being observed with compositions C4 (1% Chrysanthellum indicum,1% Cynara scolymus, 1% Vaccinium myrtillus, 0.1% piperine) and C9 (1%Chrysanthellum indicum, 1% Cynara scolymus, 1% Vaccinium myrtillus,0.001% piperine, 0.6% Olea europaea).

Composition C6 (1% Chrysanthellum indicum, 1% Cynara scolymus, 1%Vaccinium myrtillus) had no effect on the fasting glycemia. The additionof piperine (0.1%), although it (C5) induced no significant decrease inthe fasting glycemia, to the C6 combination to obtain the C4combination, made it possible, surprisingly, to significantly andstrongly lower the fasting glycemia. Similarly, the addition of Oleaeuropaea (0.6%; C8) to the C7 combination (1% Chrysanthellum indicum, 1%Cynara scolymus, 1% Vaccinium myrtillus, 0.001% piperine) to obtaincomposition C9 also made it possible to strongly lower the glycemia to alevel virtually similar to that of nondiabetic mice.

Carbohydrate Tolerance

TABLE 11 Effect of the compositions on the carbohydrate tolerance aftereight weeks of treatment AUC (week 8 − week 0, in mg × min/dL)Compositions (mean ± SEM) ad libitum control 10 954 ± 2000   Per Fedcontrol 20 355 ± 6554   C1 3367 ± 3039 * C2 1901 ± 3949 * C3 −2418 ±3260 ** C4 −11 537 ± 4168 **  C5 3610 ± 2086 * C6 13 068 ± 3064   C78639 ± 2844  C8 −8889 ± 5436 ** C9 −24 568 ± 10 440 ** Mean values ±SEM. ad libitum control, n = 22; Per Fed control, n = 10; C1, n = 9; C2,n = 8; C3, n = 10; C4, n = 8; C5, n = 9; C6, n = 14; C7, n = 12; C8, n =10; C9, n = 9. AUC: “area under the curve”. Composition versus control,Student's t test for unpaired data, * p < 0.05, ** p < 0.01.

Compositions C1, C2, C3, C4, C5, C8 and C9 brought about a decrease infeed intake. Consequently, a Per Fed group consuming the same amount offood was made. The results show that the calorie restriction imposed onthe animals had no effect on the carbohydrate tolerance (comparison ofad libitum control versus Per Fed control). Compositions C1, C2, C3, C4,C5, C8 and C9 induced a significant decrease in the AUC, which reflectsan improvement in the carbohydrate tolerance. The most pronounced effectis obtained with the combinations C4 and C9.

Body Mass

TABLE 12 Effect of the compositions on the body mass after eight weeksBody mass Compositions (in g, mean ± SEM) ad libitum control 36.9 ± 1.2Per Fed control 30.6 ± 1.2 C1 33.9 ± 2.4 C2  37.4 ± 2.2 * C3  37.0 ±2.7 * C4  36.3 ± 1.7 * C5 32.7 ± 1.5 C6    29.3 ± 1.8 *** C7 34.5 ± 1.6C8 26.9 ± 2.0 C9  25.0 ± 2.1 * Mean values ± SEM. ad libitum control, n= 34; Per Fed control, n = 10; C1, n = 9; C2, n = 9; C3, n = 9; C4, n =8; C5, n = 9; C6, n = 14; C7, n = 12; C8, n = 10; C9, n = 9. Compositionversus control, Student's t test for unpaired data, * p < 0.05, ** p <0.01, *** p < 0.0001.

Compositions C1, C2, C3, C4, C5, C8 and C9 brought about a decrease infeed intake. Consequently, a Per Fed group consuming the same amount offood was made. The results show that the calorie restriction imposed onthe animals induced a significant decrease in body mass (comparison ofad libitum control versus Per Fed control, p<0.05). Compositions C2, C3,C4, C6 and C9 also induced a significant decrease in body mass. Whereasthe combination C6 had no effect on the fasting glycemia, it induced alarge and significant decrease in body mass, giving it advantageousproperties in the prevention and treatment of obesity. The combinationC9 had the most pronounced effect.

Synergistic Effect

Moreover, the synergistic effect was evaluated according to the methodof Colby S R described in “Calculation of the synergistic andantagonistic responses of herbicide combinations” Weeds, 1967, 15:20-22.This method was especially used in patent EP03812880. For eachcombination, the synergistic factor was calculated. A factor >1indicates the existence of a synergistic effect. A factor <1 indicatesthe existence of an antagonist. The calculations performed are:

Expected degree of efficacy=A+B−(A*B/100)

Synergistic factor (SF)=(1*observed degree of efficacy (%))/expecteddegree of efficacy (%)

The calculations related to the following combinations:

-   -   Calculation of SF for the 1% Chrysanthellum indicum/1% Cynara        scolymus/1% Vaccinium myrtillus/0.1% piperine combination        (composition C4) in which A=1% Chrysanthellum indicum/1% Cynara        scolymus/1% Vaccinium myrtillus (C6) and B=piperine (C5);    -   Calculation of SF for the 1% Chrysanthellum indicum/1% Cynara        scolymus/1% Vaccinium myrtillus/0.001% piperine/0.6% Olea        europaea combination (composition C9) in which A=1%        Chrysanthellum indicum/1% Cynara scolymus/1% Vaccinium        myrtillus/0.001% piperine (composition C7) and 8=0.6% Olea        europaea (composition C8).

Table 13 below gives the results for the combination C6+C5=C4.

TABLE 13 Synergistic factors for the combination C6 + C5 = C4 ExpectedObserved degree of efficacy degree of Syner- (in % of the control group)efficacy gistic C6 C5 C4 with C4 factor Fasting glycemia +2.6% −13.3%−35.3% −11.0% 3.20 after eight weeks of treatment Oral test of +9.5%−82.3% −156.7% −82.3% 1.94 carbohydrate tolerance, AUC (week 8 − week 0)

The results presented in Table 13 are illustrated in FIGS. 2 and 3. Alarge synergistic effect with composition C4 according to the inventionis demonstrated, both on the fasting glycemia and on the carbohydratetolerance (AUC).

Table 14 below gives the results for the combination C7+C8=C9.

TABLE 14 Synergistic factors for the combination C7 + C8 = C9 ExpectedObserved degree of efficacy degree of Syner- (in % of the control group)efficacy gistic C7 C8 C9 with C9 factor Fasting glycemia +5.7% −26.1%−59.4% −21.9% 2.71 after eight weeks of treatment Oral test of +4.6%−204.5% −388.7% −209.3% 1.86 carbohydrate tolerance, AUC (week 8 − week0) Oral test −89.1% −53.2% −129.3% −94.9% 1.36 of insulin sensitivity,AUC (week 8 − week 0)

The results presented in Table 14 are illustrated in FIGS. 4, 5 and 6. Alarge synergistic effect with composition C9 according to the inventionis demonstrated, on the fasting glycemia, on the carbohydrate tolerance(AUC) and also on the insulin sensitivity (AUC). This is the case eventhough composition C7 already had a large and significant effect onimproving the insulin sensitivity.

II.2 TESTS B

Another composition, C10, was developed and tested in vivo:Chrysanthellum indicum (aerial part, dry extract)+Cynara scolymus(leaves, dry extract)+Vaccinium myrtillus (fruits, dry extract)+Pipernigrum (fruits, dry extract)+Oleo europaea (leaves, dry extract).

More precisely, composition C10 was tested in vivo in comparison withmetformin, the reference therapeutic molecule in the treatment of type 2diabetes, on the same model as Tests A (db/db mice). Using thispredictive model of diabetic mice and incorporating composition C10directly into the rodents' feed (1% Chrysanthellum indicum, 1% Cynarascolymus, 0.1% Vaccinium myrtillus, 0.001% Piper nigrum and 0.6% Oleoeuropaea of the feed), this makes it possible to ensure its“multi-target” efficacy and its large-scale use, since intravenous orintraperitoneal injections are limited to a small number of people,given their mode of administration.

The experimental time was seven weeks with a “run-in” of one weekfollowed by six weeks of supplementation with composition C10. The malemice were six weeks old at the start of the treatment. The evaluationswere made just before the supplementation (t=0), weekly for certainparameters, and at the end of supplementation (t=6 weeks).

The experimental evaluations related especially to:

-   -   Measurement of the feed intake (t1, t2, t3, t4 and t5 weeks);    -   Measurement of the body mass (t0, t1, t2, t3, t4, t5, t6 weeks);    -   Measurement of the fat mass and lean mass by MRI (magnetic        resonance imaging; t0 and t6 weeks);    -   Measurement of the fasting glycemia (t0, t1, t2, t3, t4, t5, t6        weeks);    -   Measurement of the glycated hemoglobin (HbA1c; t6 weeks);    -   The insulin sensitivity (test identical to that performed in        Tests A; t0 and t6 weeks);    -   The carbohydrate tolerance (test identical to that performed in        Tests A; t0 and t6 weeks);    -   Measurement of the hepatic and serum triglycerides (t6 weeks);    -   Measurement of the serum HDL cholesterol (t6 weeks).

These evaluations are all entirely familiar to a person skilled in theart.

The results are presented in Table 15 below. It is important to considerthat the amount of metformin ingested daily by the mice was about 26%greater than the total amount of molecules present in composition C10according to the invention. In other words, the dose of metforminadministered was greater than the dose of total molecules present incomposition C10.

Parameters Control Metformin C10 Feed intake (g/day)  9.6 ± 0.5 10.0 ±0.6 10.7 ± 0.8    Body mass (g) 44.7 ± 2.0 40.8 ± 2.1 36.3 ± 1.7 ^(a )Fat mass (%) 26.1 ± 1.5 23.2 ± 1.7 19.1 ± 1.1 ^(a ) Lean mass (%) 17.0 ±0.5 16.5 ± 0.5 16.5 ± 0.6    Fasting glycemia (mg/dL) 437 ± 26 474 ± 24160 ± 20 ^(a, b) HbA1c (%)  8.33 ± 0.53  7.59 ± 0.24  4.25 ± 0.20^(a, b) Area under the curve measured during an 50940 ± 2968 32778 ±3166 14109 ± 1856 ^(a, b) oral test of insulin sensitivity (AUC, mg ·min/dL) Area under the curve measured during an 61050 ± 2757 65614 ±1577 28823 ± 5333 ^(a, b) oral test of carbohydrate tolerance (AUC, mg ·min/dL) Serum triglycerides (mg/dL) 231.23 ± 18.29 256.23 ± 28.19 177.80± 19.60 ^(b ) Hepatic triglycerides (μmol/mg tissue) 41.10 ± 1.80 38.39± 4.84 21.39 ± 2.21 ^(a, b) HDL cholesterol (mg/dL) 74.34 ± 4.10 99.21 ±6.03 111.72 ± 8.39 ^(a  )  Mean values ± SEM. Control, n = 10;Metformin, n = 11; C10, n = 10. Student's t test for unpaired data. ^(a)C10 versus control, p < 0.05. ^(b) C10 versus Metformin, p < 0.05.

FIG. 7 more particularly illustrates the effects of metformin and ofcomposition C12 according to the invention on the insulin sensitivity.

The results illustrated in Table 15 show an extremely large andsurprising effect of composition C10 according to the invention on a setof risk factors of metabolic syndrome, of excess weight, of obesity, ofdiabetes, of non-alcoholic steatohepatitis (NASH), and of cardiovasculardiseases, namely:

-   -   The decrease in body weight via a reduction in the fat mass,        without affecting the lean mass;    -   A decrease in the fasting glycemia and in the glycated        hemoglobin;    -   An improvement in the carbohydrate tolerance;    -   An improvement in the insulin sensitivity;    -   A decrease in the hepatic and serum triglycerides;    -   An increase in the HDL cholesterol (“good cholesterol”).

Not only does the composition according to the invention decrease awhole set of risk factors, but above all it improves them to a levelvirtually equivalent to that of non-diabetic mice (healthy mice). Inother words, the invention prevents the installation and progression oftype 2 diabetes.

FIGS. 8 and 9 illustrate the effects of composition C10 according to theinvention on the change in body mass and in fasting glycemia comparedwith the control group. Composition C10 according to the inventionsubstantially limits a gain in mass and prevents an increase inglycemia.

Furthermore, its effects are very much superior to those of metformin,the main antidiabetic medicament (Ferrannini E et al. Eur Heart J 2015;Jun. 10; example: GLUCOPHAGE®) and demonstrates the absence of sideeffects on the circulating fats, in contrast with OCA (obeticholic acid,an FXR agonist intended for treating type 2 diabetes and NASH(non-alcoholic steatohepatitis)) developed by the company INTERCEPT,which drastically reduces the circulating HDL cholesterol (“goodcholesterol”) (−0.2 g/L, p<0.01). Quite to the contrary, the compositionaccording to the invention increases the level of HDL cholesterol by50.3%.

Moreover, the effect of the composition according to the invention isgreater than that of the candidate medicament developed by the companyGENFIT, GFT 505 (Elafibranor), on glycated hemoglobin (HbA1c), the maindiagnostic indicator of type 2 diabetes and of its morbid complications:composition C10, HbA1c=4% versus GFT 505, HbA1c=6% for the highest dose(same model of db/db mice and similar experimental design; Hanf R et al.Diab Vasc Dis Res 2014; 11:440-7). A further 1% decrease in HbA1creduces the risks of infarction by 14%, of cardiovascular diseases by37%, and of amputations by 43%.

Moreover, the composition according to the invention retards, or evenstops, the degeneration of the pancreatic beta cells, which are thecells responsible for insulin secretion in response to an increase inglycemia. Specifically, type 2 diabetes gradually induces a degradationof the pancreatic beta cells partly associated with chronichyperglycemia. This is reflected by an increase in insulin secretion inthe first stages of the disease to reduce the glycemia (increase ininsulinemia), followed by a gradual decrease in its secretion (lowercirculating insulinemia) due to the gradual destruction of the betacells (Leahy J L et al. J Clin Invest 1985; 77:908-915). The resultsobtained strongly support an effect of composition C10 on the pancreaticbeta cells. Specifically, after six weeks of supplementation, theinsulinemia is higher in the composition C10 group according to theinvention versus control (27.40±4.11 versus 9.64±2.53 ng/mL, p<0.01,respectively), reflecting a very different stage of advancement of thediabetes: advanced diabetes in the Control group with destruction of thebeta cells (low insulinemia, high fasting glycemia, high HbA1c) andsparingly advanced diabetes or even no diabetes in the group ofcomposition C10 according to the invention with functional beta cells(high insulinemia, low glycemia, low HbA1c).

Thus, the invention fully satisfies the need for novel preventivesolutions and therapeutic solutions taking into account the multifactornature of cardio-metabolic disorders. This invention represents a majoradvance for considerably eradicating the development of metabolicdiseases and their morbid nature.

Molecular biology measurements on the liver were also taken. Theseevaluations were performed after the six weeks of treatment and aftersacrificing the animals. In particular, the amounts of hepatic AMPK(AMP-activated protein kinase) proteins were determined by westernblotting. AMPK is considered as being a metabolic sensor. AMPK, aubiquitous enzyme, participates in the coordinated regulation of energymetabolism, food intake and the sensitivity of tissues in response tonumerous metabolic and hormonal signals. These properties thus give it arole as a major pharmacological target with metabolic applications(diabetes, insulin resistance, obesity) and cardiological applications(cardiac ischemia, diabetes-related complications) (Coughlan K A et al.Diabetes Metab Syndr Obes 2014; 7:241-53). Increasing the amounts ofAMPK constitutes a strategy of primary interest in the identificationand validation of treatments for type 2 diabetes and its complications(especially non-alcoholic steatohepatitis), and more globally forpathologies related to metabolic disorders.

The results show in the liver that composition C10 according to theinvention induces a marked increase in the amount of AMPK (control n=9,1.00±0.22 versus C10 n=9, 1.96±0.73, p<0.001, Mann Whitney Test U). Theresults are illustrated by the western blot FIG. 10.

The experimental protocol that may be performed by any person skilled inthe art is indicated in the lines below.

→AMPK

Protein Extraction

50 mg of frozen tissue (liver) were placed in 20 volumes of NP-40 buffer(50 mM Tris HCl, pH: 7.4, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1% NonidetP-40, 0.25% sodium deoxycholate) in the presence of 1 μL of proteaseinhibitor cocktail (P8340, Sigma-Aldrich) and phosphatase-inhibitingtablets (#88667 Thermo Fisher Scientific, USA). The tissues werehomogenized in ice using a glass Potter grinder and then centrifuged at14 000 g for 10 minutes at 4° C. before recovering the supernatent. Theprotein content of the supernatent was assayed with a Bio-Rad DC kit(Bio-Rad, USA) and then all the samples were brought to the samestandard concentration before being diluted a second time in Laemmli 2×buffer and then heated at 90° C. for 3 minutes.

Blotting

For each test, a molecular weight scale and an internal control weredeposited next to 15 μg of each sample on a polyacrylamide gradient gel(4-15% Mini-PROTEAN® TGX Stain-Free™ Gel, BioRad, USA). The gels werethen subjected to a current of 300 V for 18 to 20 minutes in anelectrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) beforetransferring the proteins onto PVDF membrane by means of a semi-liquidtransfer system (Transblot, Bio-Rad, USA) with a direct current of 25 Vand 2.5 A for 7 minutes. The membranes were then incubated in aTween-Tris saline buffer (TTBS: 50 mM Tris base pH: 7.5, 150 mM NaCl,and 0.01% Tween 20) enriched with 5% of BSA for 1 hour at roomtemperature. The membranes were then rinsed with TTBS and then incubatedovernight at 4° C. with anti-AMPKα antibodies (D63G4, Cell Signaling,USA). After the incubation, the membranes were washed again with TTBSand then exposed to an anti-rabbit secondary antibody conjugated tohorseradish peroxidase, at a concentration of 1:3000^(th) for one hourat room temperature. The membranes were then washed three times in TTBSbefore being exposed to a chemiluminescence solution (Clarity WesternECL; Bio-Rad, USA) for one minute. The membranes were then scanned on aBio-Rad Chemidoc system and the intensity of the bands measured usingthe supplied image analysis software (ImageLab V4.1, Bio-Rad, USA).

II.3 TEST C

One of the main drawbacks of many therapeutic molecules currently on themarket is their acute hypoglycemiant effect, with a high risk ofhypoglycemia. To test the acute hypoglycemiant effect of composition C10according to the invention, the composition was administered byforce-feeding (400 mg/kg) to six healthy C57BL/6N mice. The glycemia wasmeasured by biopsy in the tail before and after force-feeding (to, t15minutes, t30 minutes). Moreover, according to a crossed protocol, thesame mice received by force-feeding a saline solution with glycemiameasurements (to, t15 minutes, t30 minutes). The results illustrated inFIG. 11 demonstrate the absence of acute hypoglycemiant effect ofcomposition C10 according to the invention.

II.4 TEST D

A composition C11 of a single extract (aqueous-alcoholic dry extract)obtained from powder of Chrysanthellum indicum aerial parts, of Cynarascolymus leaves, of Vaccinium myrtillus fruit, of Piper nigrum fruit andof Oleo europaea leaves (ratio 1/1/0.1/0.0001/0.6) was tested on thesame model of diabetic mice used in tests A and B.

The experimental time was 6 weeks with a “run-in” of one week followedby five weeks of supplementation with composition C11. The male micewere six weeks old at the start of the treatment. The evaluation of bodymass was performed just before supplementation (t=0), weekly, and at theend of supplementation (t=5 weeks). The composition was incorporatedinto the rodents' feed at a rate of 2.7% of the food (same amount ascomposition C10).

FIG. 12 illustrates the effects of composition C11 according to theinvention on the change in body mass compared with the control group.Composition C11 according to the invention greatly limits the gain inbody mass.

III. IN VITRO EVALUATION OF THE EFFICACY OF THE COMPOSITION

in vitro experiments were also performed to demonstrate the effects ofcompositions C10 and C11 according to the invention. Composition C12 ofcombination of molecules according to the invention was also tested:dicaffeoylquinic acid+apigenin 7-O-glucuronide+monocaffeoylquinicacid+oleuropein+piperine (ratio 1/1/1/1/0.001).

Various enzymatic activity tests were performed in order to determinewhether the compositions according to the invention inhibited strategicenzymes of carbohydrate and fat metabolism: Alpha-glucosidase, DPP-IVand HMG-CoA reductase. An action on all of these strategic targetsallows global care management of type 2 diabetes and of itscomplications (diabetic foot, retinopathy, loss of vision, nephropathy,cardiovascular events, non-alcoholic steatohepatitis or NASH).

→HMG-CoA Reductase

To control the circulating fats, the objective is to reduce theconcentration of serum cholesterol by mainly targeting the inhibition ofcholesterol biosynthesis (inhibition of3-hydroxy-3-methyl-glutaryl-coenzyme A or HMG-CoA reductase).Pravastatin is one of the reference molecules for reducing LDLcholesterol and total cholesterol. It is especially sold under the nameELISOR®, PRAVASTATINE MYLAN, VASTEN®, PRAVASTATINE TEVA.

→Alpha-Glucosidase

alpha-Glucosidase is an enzyme which catalyzes the final step in theprocess of digestion of carbohydrates. alpha-Glucosidase inhibitors(examples of medicaments included in this category: GLUCOR®, DIASTABOL®)are used for reducing postprandial glycemia, which is considered as arisk factor independent of the macrovascular complications in diabetes(Kim J S et al. Biosci Biotechnol Biochem 2000; 64:2458-61).

→DPP-IV (Dipeptidyl Peptidase-IV)

DPP-IV inhibitors are considered as one of the most promising strategiesfor the care management of type 2 diabetes (von Geldern T W et al. DrugDevelopment Research 2006; 67:627-42). DPP-IV rapidly deactivates thehormones GLP-1 (glucagon-like peptide 1) and GIP (glucose-dependentinsulinotropic polypeptide), also known as incretins. Inhibition ofDPP-IV makes it possible especially to increase the action of GLP-1 andof GIP, thus allowing a physiological stimulation of insulin secretionand an inhibition of glucagon secretion by the pancreas, andconsequently a decrease in glycemia. Moreover, the inhibition of DPP-IVpromotes gastric emptying and brings about a central anorexigeniceffect. Inhibition of DPP-IV thus allows better regulation ofcarbohydrates in the case of diabetic individuals. Clinical studiesmoreover show that DPP-IV inhibitors have good efficacy with goodtolerability in the care management of hyperglycemia in the case ofindividuals suffering from type 2 diabetes, without weight gain andhypoglycemia phenomena (Green B D et al. Diabetes Vasc Dis Res 2006;3:159-65). One of the reference inhibitors is diprotin A.

The experimental protocol for evaluating the inhibitory potential ofcompositions C10, C11 and C12 was as follows:

Preparation of the Extracts, Mixtures of Extracts and Mixture of PureSynthetic Molecules

For the tests of inhibition of HMG-CoA reductase and of glucosidases,the plant extracts were diluted in ultra-pure water/ethanol mixtures,the proportions of which varied according to the type of extract, so asto obtain homogeneous solutions.

After homogenization, the samples were centrifuged at 4000 rpm for 10minutes and the supernatents were recovered (1-15 Fisher BioblockScientif centrifuge, Sigma-Aldrich®). The molecules were diluted eitherin 90/10 (v/v) ultra-pure water/DMSO or in 50/50 (v/v) ultra-purewater/ethanol.

The mixtures of several plant extracts were diluted in 50/50 (v/v)water/ethanol and were then ultrasonicated for 15 minutes, so as tohomogenize the medium (use of a UP50H Hielscher® ultrasonication probeat maximum power). Finally, they were centrifuged at 4000 rpm for 10minutes and the supernatents were recovered (1-15 Fisher BioblockScientif centrifuge, Sigma Aldrich®).

In the case of the DPP-IV test, the samples were diluted either inTrisma-HCl buffer (pH 8.0; 100 mM) or in 90/10 (v/v) ultra-purewater/DMSO.

Test of Inhibition of HMG-CoA Reductase

This test makes it possible to measure, by a decrease in absorption overtime, at λ=340 nm, the efficacy of inhibition of the enzymatic activity.The reaction mechanism is as follows:

HMG-CoA+2NADPH+2H⁺→mevalonate+2NADP⁺+CoA-SH

Disappearance of the substrate NADPH (absorbing at 340 nm) is thusmonitored over time. A decrease in the variation of the absorbance at340 nm over time relative to the control reaction without inhibitor(100% activity) is evidence of inhibition of the enzyme.

In a first stage, the solutions need to be prepared from the Sigma® kit:

-   -   the 5× buffer supplied must be diluted to a concentration of 1×        in ultra-pure water;    -   the NADPH is dissolved by adding 1× buffer, and then distributed        in 100 μL tubes;    -   the starting enzyme solution is distributed in 25 μL aliquots.

The buffer and the NADPH were stored at −20° C. and the enzyme aliquotswere stored at −80° C. until the time of use.

The manipulations were performed directly in 96-well microplates (NUNC96or SARSTEDT96). The buffers, substrates, inhibitors and the enzyme wereadded in a specific order (see below). For each microplate, it wasnecessary to prepare a blank, a control and various tests were prepared.Each element was prepared in duplicate (or even in triplicate):

-   -   preparation of the blank: 24 μL of the solvent used for the        samples, 12 μL of HMG-CoA, 158 μL of 1× buffer, 4 μL of NADPH;        incubation at 37° C., 3 to 5 min;    -   preparation of the negative control: 24 μL of the solvent used        for the samples, 12 μL of HMG-CoA, 158 μL of 1× buffer, 4 μL of        NADPH; incubation at 37° C., 3 to 5 min; addition of 2 μL of        enzyme;    -   preparation of the test: 24 μL of the potentially inhibiting        sample, 12 μL of HMG-CoA, 158 μL of 1× buffer, 4 μL of NADPH;        incubation at 37° C., 3 to 5 min; addition of 2 μl of enzyme.

After preparing the plate, the kinetic monitoring was performed in amicroplate reader (BMG Labtech) over 16 minutes, 25 seconds, with valuestaken every 25 seconds at λ=340 nm, i.e. a total of 40 cycles. Fromthis, the percentages of inhibition could be calculated.

Test of Inhibition of α-Glucosidases

The α-glucosidase inhibition tests were performed by monitoring byspectrophotometry, at λ=405 nm, the formation of the yellow-coloredproduct para-nitrophenol (PNP) from the synthetic substrate:para--nitrophenyl-α-D-glucopyranoside (PNPg), according to the followinghydrolysis reaction, catalyzed by α-glucosidases:

PNPg+H₂O→PNP+glucose

A reduction in the variation of the absorbance, at λ=405 nm, over timerelative to the control reaction without inhibitor (100% activity) isevidence of inhibition of the enzyme. The recombinant human enzymeproduced in Saccharomyces cerevisiae (maltase) (Sigma-Aldrich®, G0660)was diluted in phosphate buffer (0.1 M, pH 6.8) to pass from a stocksolution theoretically at 120 U/mL to a solution with an activity of 1.6U/mL, used for the tests.

During the α-glucosidase inhibition tests, the manipulations wereperformed directly in 96-well microplates (NUNC96 or SARSTEDT96). Thebuffers, substrates, inhibitors and the enzyme are added in a specificorder (see below). For each microplate prepared, a blank, a control andvarious tests are prepared. Each element is prepared in duplicate (oreven in triplicate):

-   -   preparation of the blank: 100 μL of phosphate buffer (0.1 M, pH        6.8), 20 μL of the solvent used for the samples, incubation at        37° C., 10 to 15 min, followed by addition of 20 μL of PNPg at        2.5 mM (dissolved in phosphate buffer);    -   preparation of the negative control: 100 μL of phosphate buffer        (0.1 M, pH 6.8), 20 μL of the solvent used for the samples, 20        μL of enzymes at 1.6 U/mL, incubation at 37° C., 10 to 15 min,        followed by addition of 20 μL of PNPg at 2.5 mM (dissolved in        phosphate buffer);    -   preparation of the test: 100 μL of phosphate buffer (0.1 M, pH        6.8), 20 μL of potentially inhibiting sample, 20 μL of enzyme at        1.6 U/mL, incubation at 37° C., 10 to 15 min, followed by        addition of 20 μL of PNPg at 2.5 mM (dissolved in phosphate        buffer).

After preparation of the plate, the kinetic monitoring was performed ina microplate reader (BMG Labtech) over 30 minutes, with values takenevery 2 minutes at λ=405 nm, i.e. a total of 16 cycles. From this, thepercentages of inhibition could be calculated.

Test of Inhibition of DPP-IV

The DPP-IV inhibition tests are performed by monitoring byspectrophotometry, at 385 nm, the formation of the productp-nitroaniline from the synthetic substrate: Gly-L-Pro-p-nitroanilide,according to the following hydrolysis reaction catalyzed by DPP-IV:

Gly-L-Pro-p-nitroanilide+H₂O→p-nitroaniline+Gly-L-pro

A decrease in the variation of the absorbance at 385 nm over timerelative to the control reaction without inhibitor (100% activity) isevidence of inhibition of the enzyme. The enzyme used was at aconcentration of 0.045 U/mL (with U=μmol/min), and it thus needed to bediluted. Since the dilution was large, it was necessary to perform twosuccessive dilutions.First dilution (production of a solution at 5×10⁻⁴ U/mL):

${D\; 1} = {\frac{5 \times 10^{- 4}*450}{0.045} = {5\mspace{14mu} {µL}}}$

(Vfinal: 450 μL; V (stock solution of enzyme)=5 μL)Second dilution (production of a solution at 5×10⁻⁶ U/mL)

${D\; 2} = {\frac{5 \times 10^{- 6}*1000}{5 \times 10^{- 4}} = {10\mspace{14mu} {µL}}}$

(Vfinal: 1000 μL; V (D1)=10 μL)→solution D2 was the usable solution.

The manipulations were performed directly in 96-well microplates (NUNC96or SARSTEDT96). The buffers, substrates, inhibitors and the enzyme wereadded in a specific order (see below). For each microplate prepared, ablank, a control and various tests were performed. Each element wasprepared in duplicate (or even in triplicate):

-   -   preparation of the blank: 25 μL of the sample, 25 μL of        substrate (Gly-L-pro-p-nitroanilide dissolved in Tris-HCl buffer        (100 mM, pH 8.0) at a concentration of 1.6 mM);    -   preparation of the negative control: 25 μL of the diluent used        for the sample, 25 μL of substrate, incubation at 37° C., 10        min, 50 μL of enzyme at 5×10⁻⁶ U/mL (prepared in 100 mM TrisHCl        buffer, pH 8.0);    -   preparation of the test: 25 μL of potentially inhibiting sample,        25 μL of substrate, incubation at 37° C., 10 min, 50 μL of        enzyme at 5×10⁻⁶ U/mL.

After preparation of the plate, the kinetic monitoring was performed ina microplate reader (BMG Labtech) over 30 minutes, with values takenevery minute at λ=385 nm, i.e. a total of 31 cycles. From this, thepercentages of inhibition could be calculated.

Calculation of the Inhibition Percentages

In general, irrespective of the test, for a given concentration of aninhibitor, a curve of equation: absorbance=f(time) (in minutes) wasprepared. Similarly, controls (without inhibitor) and blanks (withoutenzymes) were systematically prepared.

On the various curves obtained, only the linear parts at the start ofkinetics were retained, since they corresponded to the conditions fordetermining the initial rates. Thus, the slope in absorbance/minute wasproportional to the rate of appearance of the product (glucosidase andDPP-IV) or of disappearance of the substrate (HMG-CoA reductase,negative slope).

Depending on the types of tests, the percentages of inhibition of thevarious enzymes were calculated via various methods:

-   -   for the tests for inhibition of HMG-CoA reductase and of        α-glucosidases, the calculation formula is identical:

${\% \mspace{14mu} {inhibition}} = {\frac{{{control}\mspace{14mu} {slope}} - {{sample}\mspace{14mu} {slope}}}{{control}\mspace{14mu} {slope}}*100.}$

-   -   for the test of inhibition of DPP4, the formula is slightly        different since blanks are taken into account:

${\% \mspace{14mu} {inhibition}} = {\frac{\left( {{{control}\mspace{14mu} {slope}} - {{blank}\mspace{14mu} {slope}}} \right) - \left( {{{control}\mspace{14mu} {slope}} - {{blank}\mspace{14mu} {slope}}} \right)}{\left( {{{control}\mspace{14mu} {slope}} - {{blank}\mspace{14mu} {slope}}} \right)}*100.}$

Finally, a mean value of percentage inhibition was calculated for eachconcentration of inhibitor, and also the standard deviation of eachvalue. If the standard deviation values were greater than 10%, new testsin duplicate were performed, until values of less than 10% wereobtained.

The results obtained on the three enzymes for compositions C10 and C11according to the invention are summarized in Table 16 below.

% of inhibition HMG-CoA reductase ^(c) IC 50 ^(a) (μg/mL in well)(Catalytic domain of Alpha-glucosidase DPP-IV ^(b) the human enzyme −(Human recombinant (DPP-IV human recombinant GST fusion Saccharomycesrecombinant expressed protein expressed Product Cerevisiae) in SF9cells) in E. Coli) Acarbose 86.3 Absence of inhibition Absence ofinhibition Diprotin A Absence of inhibition  100% Absence of inhibitionPravastatin Absence of inhibition Absence of inhibition  100%Composition C10 4.3 15.3% 48.5% Composition C11 4.9 29.3% 32.5% ^(a) IC50: inhibitory concentration for which the enzyme has only 50% of itsactivity. ^(b) Concentration of sample set at 1.25 mg/mL. ^(c)Concentration of sample set at 60 μg/mL.

The results obtained on the three enzymes for the combination ofmolecules (composition C12 according to the invention) are summarized inTable 17 below.

% of inhibition HMG-CoA reductase ^(c) IC 50 ^(a) (μg/mL in well)(Catalytic domain of Alpha-glucosidase DPP-IV ^(b) the human enzyme −(Human recombinant (DPP-IV human recombinant GST fusion Saccharomycesrecombinant expressed protein expressed Product Cerevisiae) in SF9cells) in E. Coli) Acarbose 86.3 Absence of inhibition Absence ofinhibition Diprotin A Absence of inhibition 89.3% Absence of inhibitionPravastatin Absence of inhibition Absence of inhibition 100 CompositionC12 19.7 10.3% 30.1% ^(a) IC 50: inhibitory concentration for which theenzyme has only 50% of its activity. ^(b) Concentration of sample set at250 μg/mL. ^(c) Concentration of sample set at 60 μg/mL.

It is found that, in contrast with the reference inhibitory moleculesfor each enzyme [acarbose (medicament), diprotin A (referenceinhibitor), pravastatin (medicament) respectively], compositions C10,C11 and C12 according to the invention are, surprisingly, the only onesto inhibit the three enzymes.

By acting simultaneously on several regulation processes, thecompositions according to the invention represent an advantageouspreventive means and therapeutic means for preventing and treatingdiabetes, dyslipidemias and complications thereof. The currenttherapeutic strategies consist in combining several medicaments in orderto reduce the various risk factors individually. Nevertheless, the drugcombination may occasionally give rise to serious side reactions, forinstance the simultaneous administration of fibrates and statins whichincreases the risk of myopathy (Denke M A J Manag Care Pharm 2003;9:17-9). There is therefore at the present time a real need forpreventive solutions and medicaments whose “multi-target” mechanism ofaction has advantages in terms of compliance, tolerance and efficacy.Thus, the compositions according to the invention make it possible toreduce the risk of cardiovascular diseases and to prevent and treat eachdysfunction and/or its consequences taken independently.

What is claimed is:
 1. A composition consisting at least of: an extractobtained from a blend of at least Chrysanthellum indicum, Cynarascolymus, Vaccinium myrtillus, Piper and Olea europaea, and/or a mixtureof: an extract obtained from a blend of at least Chrysanthellum indicum,Cynara scolymus, Vaccinium myrtillus and Olea europaea, and pipérine,and/or a mixture of: an extract obtained from a blend of at leastChrysanthellum indicum, Cynara scolymus, Vaccinium myrtillus and Oleaeuropaea, and an extract of Piper containing piperine.
 2. Thecomposition of claim 1, wherein the Piper plant is chosen from Pipernigrum, Piper aduncum and Piper longum.
 3. The composition of claim 1,wherein the blend comprises Chrysanthellum indicum whole plant and/oraerial parts.
 4. The composition of claim 1, wherein the blend comprisesCynara scolymus whole plant and/or leaves.
 5. The composition of claim1, wherein the blend comprises Vaccinium myrtillus whole plant and/orfruit.
 6. The composition of claim 1, wherein the composition comprisesa mixture of the following molecules: at least one molecule chosen fromapigenin 7-O-glucuronide, chrysanthellin A, chrysanthellin B, caffeicacid, luteolin, maritimetin, eriodictyol, isookanin, apigenin, luteolin7-O-glucoside, maritimein, marein, eriodictyol 7-O-glucoside,flavomarein, apigenin 8-C-α-L-arabinoside-6-C-β-D-glucoside(shaftoside), apigenin 6,8-C-di-β-D-glucopyranoside (vicenin-2), oranalogs thereof, at least one molecule chosen from a dicaffeoylquinicacid, a sulfo-monocaffeoylquinic acid, luteolin, luteolin 7-O-glucoside,luteolin 7-O-glucuronide, apigenin 7-O-glucoside, cynaropicrin, oranalogs thereof, at least one molecule chosen from a monocaffeoylquinicacid, delphinidin 3-galactoside, delphinidin 3-glucoside, cyanidin3-galactoside, delphinidin 3-arabinoside, cyanidin 3-glucoside,petunidin 3-galactoside, cyanidin 3-arabinoside, petunidin 3-glucoside,peonidin 3-galactoside, petunidin 3-arabinoside, peonidin 3-glucoside,malvidin 3-galactoside, malvidin 3-glucoside, malvidin 3-arabinoside, oranalogs thereof, and at least piperine.
 7. The composition of claim 6,wherein the composition also comprises at least one molecule chosen fromoleuropein, hydroxytyrosol, and analogs thereof.
 8. The composition ofclaim 1, wherein the composition also comprises at least one additionalelement added to the mixture of molecules, said additional element beingchosen from: the following vitamins: B1, B2, B3, B5, B6, B8, B9, B12 C,A, D, E, K1 and K2; the following compounds: obeticholic acid, corosolicacid, polyunsaturated fatty acids of the omega 6 and/or omega 3 family,orotic acid, pangamic acid, para-aminobenzoic acid, amygdalin,beta-glucans, carnitine, dimethylglycine, imeglimin, isoflavones,L-arginine, oxytocin, pectin, pyridoxamine, resveratrol, viniferin,L-citrulline; the following trace elements and minerals: arsenic, boron,calcium, copper, iron, fluorine, iodine, lithium, manganese, magnesium,molybdenum, nickel, phosphorus, selenium, vanadium, zinc; the followingmicroconstituents of non-essential nature: conjugated linolenic acid,lipoic acid, carotenoids, carnitine, choline, coenzyme Q10,phytosterols, polyphenols of the tannin and lignan family, taurine;fructo-oligosaccharides, galacto-oligosaccharides; lacticacid-fermenting bacteria; yeasts, for example red rice yeast (Monascuspurpureus); mushroom, for example maitake; products derived from insectsthat are compatible with the food and pharmaceutical sector; marijuanaand haschisch; coating agents: for example hypromellose,microcrystalline cellulose, stearic acid, talc, sucrose, shellac,povidone, beeswax; flavors: for example natural flavor of blueberry ornatural flavor of strawberry; acidifying agents such as malic acid;antiagglomerating agents: for example silicon dioxide or magnesiumstearate; thickeners such as xanthan gum, colloidal silica, fatty acidmono- and diglycerides; stabilizers such as calcium phosphate;emulsifiers such as soybean lecithin; fillers such as corn starch;excipients selected from the group consisting of microcrystallinecellulose, magnesium stearate and dicalcium phosphate.
 9. Thecomposition of claim 1, wherein the composition is in the form of apowder, gel, emulsion, or in liquid form.
 10. The composition of claim1, wherein the composition is in the form of tablets, wafer capsules,gel capsules, sticks, sachets, vials, droppers or in injectable form.11. A method of treatment for a pathological disorder in human andanimal patients, the method comprising administering to the patient thecomposition of claim
 1. 12. The composition of claim 1, wherein thecomposition is formulated as a nutrition product and/or medicament forpreventing and/or treating pathological disorders of carbohydrate and/orfat metabolism in humans or animals.
 13. A method of preventing and/orcombating type 1 and 2 diabetes and/or non-alcoholic fatty liverdiseases and/or cardiovascular pathologies and/or pathologies associatedwith insulin resistance in a patient, the method comprisingadministering to patient, the composition of claim
 21. 14. The method ofclaim 13, wherein the non-alcoholic fatty liver disease is non-alcoholicsteatohepatitis.
 15. The method of claim 13, wherein the cardiovascularpathologies are coronary cardiopathies, cerebrovascular diseases,peripheral arteriopathies, deep vein thromboses.
 16. The method of claim13, wherein the pathology associated with insulin resistance isAlzheimer's disease.
 17. The method of claim 13, wherein the methodfurther comprises administering at least one antidiabetic therapeuticagent chosen from biguanides including metformin, dipeptidylpeptidase-IV (DPP-IV) inhibitors, glucagon-like peptide-1 (GLP-1)analogs, thiazolidinediones (TZDs), sulfonylureas, rapid and slowinsulins, sodium glucose co-transporter-2 (SGLT2) inhibitors,glycosidase inhibitors (acarbose, miglitol, voglibose, peptidescontaining the alanine-proline or proline-alanine sequence), moleculesof the fibranor family such as elafibranor, or molecules targeting thenuclear receptors and especially the ROR (α, β, γ) receptors and Rev-Erb(α, β) receptors.
 18. The method of claim 11, wherein the pathologicaldisorder is dyslipidemia.
 19. The method of claim 13, wherein the methodfurther comprises administering a hypolipemiant therapeutic agent chosenfrom: statins, fibrates, nicotinic acid, ion-exchange resins,cholesterol absorption inhibitors, omega 3 polyunsaturated fatty acids,tiadenol, and FXR (Farnesoid X Receptor) nuclear receptor agonists. 20.The method of claim 11, wherein the pathological disorder is obesity andexcess weight and/or metabolic syndrome and/or pathological problems ofarterial tension.